Activating pyruvate kinase r

ABSTRACT

The compound (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrroll-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one, or a pharmaceutically acceptable salt thereof, is useful to increase the affinity of hemoglobin for oxygen. Methods and compositions for the treatment of a hemoglobinopathies are provided herein, including certain pharmaceutical compositions for activating PKR.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application claims the benefit of and priority to each of the following co-pending patent applications: U.S. patent application Ser. No. 16/576,720, filed Sep. 19, 2019; U.S. patent application Ser. No. 16/576,360, filed Sep. 19, 2019; U.S. Patent Application No. 62/902,887, filed Sep. 19, 2019; U.S. Patent Application No. 62/906,437, filed Sep. 26, 2019; International Application No. PCT/US2019/052024, filed Sep. 19, 2019; U.S. Patent Application No. 63/024,432, filed May 13, 2020; U.S. Patent Application No. 63/024,441, filed May 13, 2020; U.S. Patent Application No. 62/704,785, filed May 28, 2020; and U.S. Patent Application No. 62/705,106, filed Jun. 11, 2020; each of which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

This disclosure relates to therapeutic compounds, compositions and methods comprising the administration of compounds that activate pyruvate kinase R (PKR), including methods of treating hemoglobinopathy conditions by the administration of therapeutic compositions activating pyruvate kinase R (PK-R).

BACKGROUND

Hemoglobin is a tetrameric protein which binds oxygen in Red Blood Cells (RBC). Oxygen binds to the four hemes of the hemoglobin molecule. Each heme contains porphyrin and ferrous iron that reversibly binds oxygen through an iron-oxygen bond. Binding of each of four successive oxygen molecules to the heme requires less energy than the previous bound oxygen molecules. Hemoglobin has two alpha and two beta subunits symmetrically arranged to form dimers that rotate during oxygen release to open a central water cavity. An allosteric transition including movement of the alpha-beta dimer takes place between the binding of the third and fourth oxygen. In blood, hemoglobin is in equilibrium between two allosteric structures: a deoxygenated (tense, or “T” state), and an oxygenated (relaxed or “R” relaxed) state.

Pharmaceutical compositions for influencing the allosteric equilibrium of hemoglobin (e.g., by increasing the affinity of oxygen for hemoglobin) are useful for treating various diseases or conditions. For example, increasing the affinity of hemoglobin for oxygen can provide a variety of medical benefits, such as the treatment of Sickle Cell Anemia or other hemoglobinopathies. For example, therapeutic approaches that increase oxygen affinity (i.e., reduce deoxygenation) of HgbS would presumably decrease polymer formation, changes to the cell membrane, and clinical consequences associated with certain hemoglobinopathy conditions such as SCD.

Hemoglobinopathy is a diverse range of rare inherited genetic disorders that affect hemoglobin, the iron-containing protein in RBCs responsible for transporting oxygen in the blood. Normal hemoglobin is a tetramer of two beta-globin and two alpha-globin protein subunits. Mutations in either the beta- or alpha-globin genes may cause abnormalities in the production or structure of these subunits that can lead to toxicity to or reduced oxygen carrying capacity of RBCs. Collectively, disorders that arise from these mutations are referred to as hemoglobinopathies.

SCD is the most common type of hemoglobinopathy. SCD is a common single-gene disorder. SCD is a recessive disease caused by inheritance of hemoglobin S (HbS) a mutated form of the β-globin gene, together with another copy of HbS, or a different defective β-globin gene variant. Due to its chronic nature, the economic burden of SCD is high, both in terms of direct costs for lifelong management, hospitalizations and associated morbidities, and indirect costs of lost lifetime earnings and reduced productivity of both patients and caregivers. The current therapeutic treatment of SCD is inadequate. Acute painful VOC events are common, occurring on approximately 55% of days, as self-reported in SCD patients. Supportive care for the management of painful VOCs entails the use of opioids, which are effective at managing pain but are highly addictive. For most patients treatment involves the chronic use of hydroxyurea, or HU, an oral chemotherapy, which stimulates production of fetal hemoglobin, or HbF, and reduces sickle hemoglobin, or HbS, polymerization and consequent RBC sickling. While inducing HbF can be effective therapeutically, HU can suppress bone marrow function and cause birth defects. Although HU is considered to have an acceptable therapeutic index given the consequences of SCD, HU is underutilized due to safety concerns and side effects. Recent approval of voxelotor and crizanlizumab will evolve the treatment paradigm but are in early stages of adoption, and neither drug provides a complete solution, which is to address underlying anemia and to reduce clinical sequalae such as VOCs. FIG. 1 illustrates certain therapeutic strategies and approved modalities for the treatment of SCD.

Beta thalassemia is a rare genetic disease with an estimated prevalence of approximately 20,000 patients across the United States and Europe and approximately 300,000 patients globally. In beta thalassemia, mutations in the beta-globin gene cause production of a defective beta-globin subunit or the absence of a beta-globin, which results both in a reduction in the total amount of oxygen carrying by RBCs as well as an excess of alpha hemoglobin subunits that aggregate and cause RBC toxicity and destruction, or hemolysis. The spleen in these patients is often enlarged due to the high rate of chronic hemolysis. Chronic hemolysis leads to elevated levels of bilirubin which can form stones in the gall bladder that can cause obstruction. To compensate for the anemia in these patients, the bone marrow, the typical RBC producing tissue, expands, and RBC production outside of the bone marrow in organs such as the liver can occur. This expansion of the bone marrow can lead to bone deformities.

Given the current standard of care for SCD and beta thalassemia, there is a clear medical need for a noninvasive, disease-modifying therapy with appropriate safety and efficacy profiles. While there has been an increase in novel therapeutic approaches for the treatment of SCD, there remain limited treatment options for these patients and drugs with improved efficacy and tolerability are still needed to manage patients with this disease. Due to the progressive nature of SCD, early interventions that modify the disease but do not affect pediatric growth and development are needed. Emerging treatments for SCD target the mechanism of disease (HbS polymerization) or the downstream consequences of RBC deformation (e.g. vasoocculsion) or the underlying cause of disease (mutations in hemoglobin); however, these treatment strategies are limited in their outcomes and applicability, and disease-modifying therapies that are safe, effective and accessible for the majority of SCD patients are needed. Despite currently available treatment options, significant unmet needs remain as most patients with SCD suffer from significant morbidity, reduced quality of life, lifelong disability and average life expectancy that is 25 to 30 years lower than that of unaffected adults.

SUMMARY

The compound (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one (“Compound 1”) can be administered once per day (QD)). The pharmacological response of Compound 1 is observed for a time period sufficient to support once daily (QD) dosing, despite having an observed concentration in the blood of human subjects at a concentration below the AC₅₀ within a few hours of administration. For example, FIG. 43 shows the pharmacokinetic (PK) measurement of the blood concentration of Compound 1 in humans (circles) and the pharmacodynamic measurement of the resulting concentration of 2,3-DPG measured in these subjects (squares) after the administration of a single dose of Compound 1. The observed maximum 2,3-DPG decrease occurred about 16 to 24 hours post-dose and was sustained up to about 48 hours after administration. In addition, the observed increase in hemoglobin oxygen affinity in humans was comparable after once daily and twice daily administration of Compound 1. Compound 1 unexpectedly increased hemoglobin oxygen affinity in humans to a comparable degree in once daily and twice daily administration. FIG. 42 is a graph showing that the effect on oxygen affinity (measured as p50) measured 24 hours after administration of Compound 1 is similar with once daily and twice daily dosing. The PK profile of Compound 1 was biphasic with a terminal half-life of about 12-14 hours. Overall, the observed pharmacodynamic response in HVs was surprisingly durable, with 2,3-DPG depression observed long after plasma Cmax, with an apparent PD half-life supporting QD dosing. Accordingly, in some embodiments, methods of treatment comprise the once daily (QD) administration of Compound 1 (i.e., not twice per day or BID), or a pharmaceutically acceptable salt thereof, to a patient in need thereof, such as a patient diagnosed with a hemoglobinopathy such as Sickle Cell Disease (SCD).

Following 14 days of dosing in healthy subjects in the clinical trial of Example 12, the observed clearance on day 1 and day 14 was unchanged, providing clinical evidence that the PK of Compound 1 is time-independent and not a substrate of auto-induction or auto-inhibition at the doses tested.

One aspect of the disclosure relates to methods of treating a patient, such as a patient diagnosed with a hemoglobinopathy, comprising the administration of a therapeutically effective amount of a PKR Activating Compound or a pharmaceutically acceptable salt thereof. As used herein, a “PKR Activating Compound” is a compound having an AC₅₀ value of less than 1 micro Molar using the Luminescence Assay described in Example 2, or a pharmaceutically acceptable salt and/or other solid form thereof.

The compound (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one (“Compound 1”) is a selective, orally bioavailable PKR Activating Compound that decreases 2,3-DPG, increases ATP, and has anti-sickling effects in disease models with a wide therapeutic margin relative to preclinical toxicity.

Compound 1 is an allosteric activator of recombinant wild type (WT) PKR and a mutant enzyme, PKR R510Q which is one of the most prevalent PKR mutations in North America. PKR exists in both a dimeric and tetrameric state, but functions most efficiently as a tetramer. Pyruvate kinase R (PKR) is the isoform of pyruvate kinase expressed in RBCs, and is the rate limiting enzyme in the glycolytic pathway. Compound 1 stabilizes the tetrameric form of PKR, thereby lowering the Michaelis-Menten constant (Km) for its substrate, phosphoenolpyruvate (P).

Compound 1 can be orally administered once per day (QD) to a patient in need thereof which is a significant benefit in a patient population requiring lifelong therapy. Compound 1 was evaluated in a randomized, placebo-controlled, double blind, single ascending and multiple ascending dose study to assess the safety, pharmacokinetics, and pharmacodynamics of Compound 1 in healthy volunteers in both single ascending dose (SAD) cohorts and in multiple ascending dose (MAD) cohorts. Four healthy SAD cohorts were evaluated at doses of 200, 400, 700, and 1000 mg, and four healthy MAD cohorts received 200 to 600 mg total daily doses for 14 days at QD or BID dosing (100 mg BID, 200 mg BID, 300 mg BID, and 400 mg QD).

In some embodiments, the compound (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one (“Compound 1”) is useful in a single daily (QD) administration to increase hemoglobin oxygen affinity in the red blood cells (RBCs) of a human subject as measured by a reduced p50 (pO2 at 50% hemoglobin saturation) measured in the RBCs at 24 hours after the administration of the compound. In some embodiments, Compound 1 can be used in daily (QD) administration for 14 consecutive days to increase hemoglobin oxygen affinity in the red blood cells (RBCs) of a human subject as measured by a reduced p50 (pO2 at 50% hemoglobin saturation) measured in the RBCs at after 14 days of QD administration of the compound to the human subject. In some embodiments, Compound 1 is useful in reducing the 2,3-DPG concentration in the blood of the human subject by at least 30% at 24 hours after the administration of the compound. In some embodiments, Compound 1 is useful in increasing the ATP concentration in the blood of the human subject by at least 40% after administering the compound once daily to the subject for 14 consecutive days. In some embodiments, Compound 1 is useful in simultaneously activating PKR, increasing ATP, decreasing 2,3-DPG and increasing oxygen affinity (p50) in the blood of the subject for 72 hours after administering the compound to the subject.

In some embodiments, Compound 1 can be administered to a human subject diagnosed with Sickle Cell Disease (SCD). In some embodiments, the human subject is a pediatric SCD patient who is at least age 12. In some embodiments, the human subject is at least age 18.

In some embodiments, Compound 1 is useful in treating a human subject diagnosed one of the following hemoglobin genotypes: Hgb SS, Hgb Sβ+-thalassemia, Hgb Sβ0-thalassemia, or Hgb SC.

In some embodiments, the compound (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one for use in the treatment of Sickle Cell Disease in a human subject having a Hgb SS or Hgb SC hemoglobin genotype.

In RBCs of the healthy volunteers, Compound 1 demonstrated a reduction in 2,3-DPG and an increase in ATP. In addition, the reduction of 2,3-DPG correlated with increased oxygen affinity with single and multiple doses of Compound 1. In the SAD cohorts, the healthy subjects' maximum decreases in 2,3-DPG levels generally occurred about 24 hours after the first dose with the reduction sustained about 48-72 hr postdose. After 14 days of Compound 1 dosing these PD effects were maintained along with an increase in ATP over baseline. The healthy volunteers who received a single dose of Compound 1 experienced a decrease in p50 measured 24-hours post-dose, relative to subjects who received the placebo. In the MAD cohorts, the subjects' maximum decrease in 2,3-DPG on Day 14 was 55% from baseline (median), and the 2,3-DPG levels reached a nadir and plateaued on Day 1 and did not return to baseline levels until 72 hours after the final dose on Day 14. Healthy subjects in the MAD cohorts who received Compound 1 experienced a decrease in blood 2,3-DPG levels, relative to subjects who received the placebo. Notably, these effects were maintained for more than one day after Compound 1 dosing was stopped at day 14. In addition, p50 (PO₂ at 50% hemoglobin saturation) of healthy subjects in the MAD cohorts determined after 14 days of twice daily dosing were reduced at all dose levels tested (median reduction ranged from ˜3-5 mmHg). In addition, the MAD cohort healthy subjects' blood ATP levels measured were elevated, relative to baseline, on day 14, and (notably) remained elevated for about 60 hours and returned to baseline 72 hours after the last dose.

In healthy volunteers who received single doses of Compound 1, dose normalized Cmax and AUC increased with increasing doses ≥700 mg suggesting greater than dose proportional increases in exposure at the highest doses tested (FIG. 31A). Compound 1 exhibited dose linear increase in exposure and time-independent PK, where PK parameters (Cmax, AUC) are similar after 14 days of QD dosing (FIG. 31B) and the PD activity of Compound 1 was observed at all dose levels after 24 h (decreased 2,3-DPG, p<0.0001) and after 14-days (increased ATP, p<0.0001) of dosing. The biologic consequence of this PD response was an increase in oxygen affinity (decreased p50, p<0.0001) within 24 h of Compound 1 dosing and a decrease in absolute reticulocyte counts (p<0.0001) with a slight increase in hemoglobin levels (ns) by Day 4 of the dosing period in all Compound 1 dose cohorts. Administration of Compound 1 for 3 days reduced reticulocytes (p<0.0001), along with increased hemoglobin (ns). Decreased reticulocyte counts may reflect increased RBC lifespan in healthy volunteers.

Applicant has also discovered that the increase in oxygen affinity observed in subjects treated with Compound 1 correlated with the reduction of 2,3-DPG. That is, the observed decrease in 2,3-DPG (the independent variable) after the administration of Compound 1 was correlated with an observed increase in oxygen affinity (the dependent variable) in humans receiving Compound 1 in the clinical trial of Example 12. A positive correlative relationship between 2,3 DPG and p50 levels was observed for healthy subjects receiving various doses of Compound 1 in the SAD and MAD cohorts: the increase in oxygen affinity in subjects treated with Compound 1 correlated with the reduction of 2,3-DPG. However, the observed 2,3 DPG modulation does not track directly plasma pharmacokinetics (blood concentration of Compound 1) for healthy subjects after administration of a single dose of Compound 1 (400 mg), where the pharmacodynamic maximum (i.e., the minimum of the 2,3-DPG concentration, at time ˜24 h) occurred nearly 24 h after the Cmax (i.e., maximum of the PK curve, at time ˜1-2 h).

Compound 1 was evaluated in a randomized, placebo-controlled, double blind, single ascending and multiple ascending dose study to assess the safety, pharmacokinetics, and pharmacodynamics of Compound 1 in sickle cell disease (SCD) patients. Compound 1 was well tolerated and has favorable biologic effects in SCD patients tested, with evidence of pharmacodynamic activity translating into increased oxygen affinity, a shift in the Point of Sickling to lower oxygen tensions, and improved membrane deformability of sickle RBCs at low values of pO₂ compared to pre-treatment baseline values. Based on the safety and PK/PD profile in healthy volunteer studies, a single 700 mg single dose was initially evaluated in patients with SCD (n=7). All patients had a Hb SS genotype and a mild VOC history but persistent anemia and ongoing hemolysis, despite hydroxyurea therapy.

Increased hemoglobin O₂ affinity (decreased p50) was observed after a single 700 mg dose of Compound 1 in patients with SCD, and the increased hemoglobin O₂ affinity correlated with a reduction in 2,3-DPG in patients with SCD. The maximum 2,3-DPG and ATP responses were observed 24 hours after administration of Compound 1. A single dose of Compound 1 resulted in an increase in Hb of 0.5 g/dL (range: 0.3, 0.9) in Compound 1-treated participants vs. a decrease in Hb of 0.4 g/dL (range: −0.5, −0.3) in placebo-treated participants (decreased Hb potentially due to phlebotomy). The decrease in Hb in placebo patients was attributed to phlebotomy performed to obtain blood for PK/PD measurements over the first 24 hour period. Thus, there was a mean Hb difference of ˜0.9 g/dL in participants receiving Compound 1 or placebo. Decreased lactate dehydrogenase (LDH) was also observed in Compound 1-treated participants 72 hours after Compound 1 dosing, indicating a reduction in RBC hemolysis. Compound 1 decreased the point of sickling (the partial pressure of O₂ at which HbS polymerization causes stiffening of the RBC) and improved sickle RBC O₂-dependent deformability, as demonstrated by an increase in the minimum elongation index (EI_(min)) measured in the Oxygenscan. Compound 1 increased 02 affinity (decreased p50) in all participants treated. Compound 1 improved osmolality-dependent membrane function in sickle RBCs, as demonstrated by improvements (i.e., right shifts toward normal) in O_(min) and O_(hyper) measured with Osmoscan. Osmoscan evaluates RBC membrane function (deformability) across an osmolality gradient. The Osmoscan of SCD RBCs is differentiated from that obtained from healthy RBCs in the following ways: (1) the O_(min) is reduced (shifted to the left), reflecting an increased surface/volume ratio, (2) the ratio of EI_(max)/O_(max) is reduced (shifted to the left) reflecting reduced deformability and poor ion channel function, and (3) the O_(hyper) is reduced (shifted to the left), reflecting increased RBC viscosity and decreased RBC cell volume. These effects were transient, returning to baseline 3 to 7 days after the single dose of Compound 1. SCD subjects who received a single dose of Compound 1 experienced increased oxygen affinity of HbS, attaining an oxygen dissociation curve similar to HbA, and also experienced a left shift in the point of sickling (PoS) with an increase in the EImin.

Compound 1 improved oxygen affinity, decreased point of sickling and improved deformability in patients diagnosed with SCD. Compound 1 also improved membrane function, demonstrated by an improved response to an osmotic gradient under shear stress. A single dose of Compound 1 resulted in improvements in hemoglobin, RBCs, and reticulocyte counts occurred when maximum PD effects were observed. These improvements indicate a sustained 2,3-DPG reduction and increased ATP production were observed after treatment with Compound 1.

Compound 1 was well-tolerated in clinical trials and has not shown evidence of inhibition of aromatase, an enzyme involved in converting testosterone to estrogen, which may permit dosing in a broad range of patients, including both pediatric and adult populations, as it does not lead to alterations in the hormones that affect pediatric growth and development. In addition, Compound 1 demonstrated a lack of cytochrome P450, or CYP, inhibition or induction, thereby reducing risk for drug-drug interactions due to CYP's effects on pharmacokinetics of other drugs through changes in plasma concentration.

In some embodiments, pharmaceutical compositions comprising Compound 1 can be formulated for use as an oral, once-daily, potentially disease-modifying therapy for the treatment of SCD. Compound 1 can modulate RBC metabolism by impacting two critical pathways through PKR activation: a decrease in 2,3 diphosphoglycerate (2,3-DPG), which increases oxygen affinity and an increase in adenosine triphosphate, or ATP, which may improve RBC and membrane health and integrity, reducing RBC hemolysis and increasing lifespan. In some embodiments, multi-modal methods of treatment can comprise the administration of Compound 1 to improve hemoglobin levels through increased RBC survival and decrease VOCs through reduced RBC sickling and hemolysis. In some methods, Compound 1 is administered to modify SCD at an early age, potentially preventing end-organ damage, reducing hospitalizations, and improving the patients' overall health and quality of life. In some embodiments, methods of treatment comprise administration of a therapeutically effective amount of Compound 1 to modulate RBC metabolism via a multi-modal approach by decreasing 2,3-DPG and increasing ATP.

Some embodiments provide an oral, once-daily dosage form (e.g., a tablet or capsule) comprising Compound 1 for use in a therapy for increasing hemoglobin oxygen affinity by reducing 2,3-DPG blood concentrations, increasing hemoglobin levels and/or increasing intracellular ATP, without significant effects on sex hormones (e.g., without aromatase inhibition activity) or inducing its own metabolism upon repeat daily administration throughout a course of treatment.

Even a single dose of Compound 1 resulted in favorable biologic effects including: (1) improved oxygen affinity, decreased point of sickling and improving deformability at low oxygen concentration, (2) improved membrane function, demonstrated by an improved response to an osmotic gradient in the presence of a shear stress, and (3) increased hemoglobin and RBCs and decreased reticulocytes when maximum PD effects were observed, indicating a sustained 2,3-DPG reduction and increased ATP production may improve the hemolytic anemia and the frequency of VOCs that characterize SCD. In addition, Compound 1 improves SCD patient RBC deformability, increases oxygen affinity and improves osmolality dependent membrane function. A single dose of Compound 1 has a favorable safety profile in patients with SCD.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of hemoglobin mutations giving rise to hemoglobinopathies summary of current therapeutic strategies for the treatment of sickle cell disease.

FIG. 2 is a pair of graphs comparing 2,3-DPG and ATP levels in SCD RBCs and healthy RBCs.

FIG. 3 is a schematic showing the relationship of PKR activation to the reduction of the clinical consequences of sickle cell disease (SCD).

FIG. 4 is a diagram of the proposed mechanism of action of Compound 1.

FIG. 5 is a diagram of hemoglobin mutations giving rise to hemoglobinopathies.

FIG. 6 is a graph showing the oxyhemoglobin dissociation curve and modulating factors by plotting the relationship between hemoglobin saturation (percent) vs. partial pressure of oxygen (mmHg).

FIG. 7 is a graph showing activation of recombinant PKR-R510Q with Compound 1, plotting the normalized rate vs. concentration of phosphoenolpyruvate (PEP) (Example 3).

FIG. 8 is a graph of data showing activation of recombinant PKR-R510Q by Compound 1 in the enzyme assay of Example 3.

FIG. 9 is a graph of data showing PKR activation in human red blood cells treated with Compound 1 (Example 4).

FIGS. 10 and 11 are graphs of data showing the effect of treatment with Compound 1 on oxyhemoglobin dissociation in RBCs from SCD patients (Example 5). FIG. 10 shows each data point in grayscale, while FIG. 11 shows the same data with stylized lines.

FIG. 12 is a graph of data showing delta curves of hemoglobin saturation at different oxygen tensions for red blood cells from SCD patients (Example 5). The measurement intervals are 1 mmHg.

FIG. 13 is a graph of data showing an effect of Compound 1 on sickling of human SCD cells under hypoxic conditions (Example 5).

FIG. 14 is a graph showing the effect of Compound 1 on the oxygen affinity on RBCs from healthy donors and SCD donors.

FIG. 15 is a graph showing the effect of Compound 1 on SCD RBC sickling.

FIG. 16 is a graph showing the effect of Compound 1 on P50 in HbS RBCs.

FIG. 17 is a graph showing the effect of Compound 1 on elongation index in HbS RBCs, as measured by oxygen scan.

FIG. 18A (Study 1) and FIG. 18B (Study 2) are each graphs showing the observed changes in 2,3-DPG levels in blood from mice following 7 days of once daily (QD) oral treatment with Compound 1 (Example 8).

FIG. 19 is a graph showing observed changes in 2,3-DPG levels in blood from mice following 7 days of once daily (QD) oral treatment with Compound 1 (Example 8, Study 2).

FIG. 20A (Study 1) and FIG. 20B (Study 2) are graphs of data measuring ATP concentrations in red blood cells of mice following 7 days of once daily (QD) oral treatment with Compound 1 (Example 8).

FIG. 21 is a graph showing the effect of Compound 1 on ATP levels in non-human primates.

FIG. 22 is a graph showing the effect of Compound 1 on 2,3-DPG levels in non-human primates.

FIG. 23A and FIG. 23B are each a graph of data showing oxygen saturation in RBCs following 7 days of oral treatment with Compound 1 in a murine model of SCD (Example 11).

FIG. 24A and FIG. 24B are each a graph of data showing change in oxygen saturation in RBCs following 7 days of oral treatment with Compound 1 in a murine model of SCD (Example 11).

FIG. 25 is a graph of data showing the percentage of sickled cells in a murine model of SCD following 7 days of oral treatment with Compound 1 (Example 11).

FIG. 26 is a graph of data showing the reticulocyte count in cells in a murine model of SCD following 7 days of oral treatment with Compound 1 (Example 11).

FIG. 27 is a graph demonstrating the 2,3-DPG and oxygen affinity of Hgb S RBCs in comparison to Hgb A RBCs.

FIG. 28 is a summary of a SAD/MAD trial to assess the safety and PK/PD of Compound 1.

FIG. 29 is a graph depicting Compound 1 plasma concentrations following a single dose of Compound 1 in healthy volunteers.

FIG. 30 is a graph of the blood 2,3-DPG levels measured over time in healthy volunteers who received a single dose of Compound 1 or placebo.

FIG. 31A is a table of data obtained from the single ascending dose (SAD) human clinical study of Compound 1 described in Example 12, showing pharmacokinetic (PK) properties of single doses of Compound 1. Values are presented as geometric mean [CV %] for Cmax, AUC₀₋₂₄, and half-life; and median [CV %] for Tmax.

FIG. 31B is a table of data obtained from the multiple ascending dose (MAD) human clinical study of Compound 1 described in Example 12, showing time-independent pharmacokinetic (PK) properties over 14 days of dosing Compound 1 either QD or BID. Values are presented as geometric mean [CV %] for Cmax, AUC_(0-tau), Ratio Day14/Day1 Cmax, and Ratio Day14/Day1 AUC_(0-tau); and median [CV %] for Tmax.

FIG. 32 is a graph of the blood 2,3-DPG levels measured 24 hours post-dose in healthy volunteers who received a single dose of Compound 1 or placebo.

FIG. 33 is a graph of the p50 values measured 24 hours post-dose in healthy volunteers who received a single dose of Compound 1 or placebo.

FIG. 34 is a graph of the p50 values measured pre-dose and 24-hours post-dose in healthy volunteers who received a single dose of Compound 1 or placebo.

FIGS. 35 and 36 are graphs of the blood 2,3-DPG levels measured over time in healthy volunteers who received daily doses of Compound 1 or placebo for 14 days.

FIG. 37 is a graph of the blood 2,3-DPG levels measured on day 14 in healthy volunteers who received daily doses of Compound 1 or placebo for 14 days.

FIG. 38 is a graph of the p50 values measured on day 14 in healthy volunteers who received daily doses of Compound 1 or placebo for 14 days.

FIG. 39 is a graph of the p50 values measured pre-dose and on day 14 in healthy volunteers who received daily doses of Compound 1 or placebo for 14 days.

FIG. 40 is a graph of the blood ATP levels measured on day 14 in healthy volunteers who received daily doses of Compound 1 or placebo for 14 days.

FIG. 41 is a graph showing the effect of Compound 1 on ATP levels in RBCs of healthy volunteers.

FIG. 42 is a graph showing the difference in the p50 values determined pre-dose and 24 hours post-dose (SAD cohorts) and 24 hours post-dose on day 14 (MAD cohorts) in healthy volunteers who received Compound 1 or placebo.

FIG. 43 is a graph plotting the blood concentration of Compound 1 (ng/mL) measured in healthy volunteer (HV) patients on a first (left) axis and the concentration of 2,3-DPG (micrograms/mL) measured in these HV patients on a second (right) axis after administration of a single dose of Compound 1 (400 mg).

FIG. 44 is a scatter plot of 2,3-DPG levels and p50 values observed in healthy volunteers in the SAD and MAD cohorts.

FIG. 45 is a scatter plot of 2,3-DPG levels and p50 values observed in subjects treated with Compound 1.

FIG. 46 is a graph depicting a model of the predicted PD response of once daily (QD) doses of Compound 1 in healthy volunteer RBCs.

FIG. 47 is a graph of the mean plasma concentration of Compound 1 over time in SCD patients and healthy volunteers following a single 700 mg dose of Compound 1.

FIG. 48 is a graph of 2,3-DPG and ATP blood concentrations over time in SCD patients following a single 700 mg dose of Compound 1 or placebo.

FIG. 49 is a graph oxygen affinity (p50) before and 24 hours after a single 700 mg dose of Compound 1 in healthy volunteer and SCD patients.

FIG. 50 is a scatter plot of 2,3-DPG levels and p50 values observed in healthy volunteers and SCD patients before and after administration of Compound 1.

FIG. 51 depicts four graphs showing changes from baseline in hematologic laboratory parameters in SCD patients following a single dose of Compound 1 or placebo.

FIG. 52 is a pair of graphs depicting the effects of a single dose of Compound 1 or placebo on oxygen scan in SCD patients.

FIG. 53 is a pair of graphs depicting the effects of a single dose of Compound 1 or placebo on oxygen affinity (PO₅₀) in SCD patients.

FIG. 54 is a pair of graphs depicting the effects of a single dose of Compound 1 or placebo on osmoscan in SCD patients.

FIG. 55A is a graph of hemoglobin oxygen saturation versus pO2 in SCD subjects before and after a single dose of Compound 1.

FIG. 55B is a graph of elongation index (EI) versus pO2 in SCD subjects before and after a single dose of Compound 1.

FIG. 56 is a summary of a phase 2/3, randomized, double-blind, placebo-controlled global study (PRAISE) to investigate the safety and efficacy of Compound 1 in patients with SCD.

FIG. 57 is a graph showing the concentration of Compound 1 administered in different compositions, measured over time measured in rats in the bioavailability experiment of Example 17.

FIG. 58 is a graph showing the exposure (compound 1 plasma concentration in ng/mL over time for 24 hours) of Compound 1 administered non-human primates in different compositions, as described in Example 17.

DETAILED DESCRIPTION

The PKR Activating Compound (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one (Compound 1):

is a selective, orally bioavailable PKR Activating Compound that decreases 2,3-DPG, increases ATP, and has anti-sickling effects in disease models with a wide therapeutic margin relative to preclinical toxicity. Compound 1 is a potent activator of PKR and a multi-modal metabolic modulator of RBCs. Activation of PKR simultaneously reduces 2,3-DPG concentrations, which increases hemoglobin-oxygen affinity and decreases sickling, while also increasing intracellular ATP, which improves RBC health and reduces hemolysis, or RBC death.

Compound 1 can be identified as a PKR Activating Compound of Formula I.

(including, e.g., Compound 1 and mixtures of Compound 1 and Compound 2) having an AC₅₀ value of less than 1 μM using the Luminescence Assay described in Example 2.

Compound 1 potentially represents an important advancement for patients living with SCD and other hemoglobinopathies, including beta thalassemia. PKR Activating Compounds, such as 1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one, or a pharmaceutically acceptable salt thereof, are useful in pharmaceutical compositions for the treatment of patients diagnosed with hemoglobinopathies such as SCD. The invention is based in part on the discovery that the activation of PKR can target both sickling, by reducing deoxy-HgbS, and hemolysis. Compound 1 decreases 2,3-DPG, increases ATP in RBCs and increases oxygen affinity of hemoglobin (as measured by a left shift in the partial pressure of oxygen at 50% hemoglobin saturation, or p50) in patients diagnosed with a hemoglobinopathy such as Sickle Cell Disease.

Compound 1 modulates RBC metabolism via a multi-modal approach by decreasing 2,3-DPG and increasing ATP. Decreasing the concentration of 2,3-DPG has been observed to normalize hemoglobin-oxygen affinity and decrease RBC sickling in vitro. Reduced RBC sickling has the potential to improve patients' hemoglobin levels and reduce their VOCs. Compound 1 may also improve RBC membrane health and integrity by increasing ATP, resulting in a more flexible RBC membrane for improved blood flow and potentially lessening the occurrences of VOCs. Improvement of RBC membrane health by increasing ATP is particularly useful in the setting of beta-thalassemia. A rapid onset of activity has been observed within hours in vitro and within 24 hours in healthy volunteers and SCD patients, including improved RBC deformability across an oxygen gradient (oxygen scan) and across an osmolality gradient (osmoscan), indicating an effect on RBC sickling and RBC membrane health, respectively. The relatively rapid onset of Compound 1's impact contrasts with current treatment regimens that applicant believes may take longer to demonstrate anti-sickling effects, improvements in Hb and RBC counts, or decreases in reticulocyte counts.

Applicant has discovered that Compound 1 may be administered orally once daily. A dose-exposure-response analysis utilizing the pharmacokinetics/pharmacodynamics, or PK/PD, of results obtained from healthy volunteers and SCD patients supports once-daily dosing, without the need for extensive monitoring or dose adjustments, potentially improving compliance issues historically seen with SCD patients.

Compound 1 Activates PKR

Pyruvate kinase R (PKR) is the isoform of pyruvate kinase expressed in RBCs, and is a key enzyme in glycolysis. PKR plays a major role as a regulator of metabolic flux through glycolysis. Activation of PKR offers the potential to decrease 2,3-DPG and increase ATP, which would reduce RBC sickling and cell membrane damage from HbS polymerization. As illustrated in FIG. 2 , 2,3-DPG levels are significantly higher and ATP levels significantly lower in SCD RBCs compared with normal healthy RBCs. Through a reduction in 2,3-DPG and an increase in ATP, a PKR activator has the potential to positively impact physiological changes that lead to the clinical pathologies of SCD and yield a broader and more significant impact on SCD disease than other agents that directly modify HbS, which may not otherwise improve RBC health and membrane integrity.

The invention is based in part on the discovery that the activation of PKR can target both sickling, by reducing deoxy-HgbS, and hemolysis. Targeting hemolysis may be achieved by improving RBC membrane integrity. One aspect of the disclosure is the recognition that activation of PKR can reduce 2,3-diphosphoglycerate (2,3-DPG), which leads to decreased deoxy-HgbS (and, therefore, sickling), as well as can increase ATP, which promotes membrane health and reduces hemolysis. Another aspect of the disclosure is the recognition that activation of PKR can reduce 2,3-diphosphoglycerate (2,3-DPG), which inhibits Hgb deoxygenation/increases oxygen affinity of HgbS and leads to decreased deoxy-HgbS (and, therefore, sickling), as well as can increase ATP, which promotes membrane health and reduces hemolysis. Accordingly, in one embodiment, PKR activation (e.g., by administration of a therapeutically effective amount of a PKR Activating Compound to a patient diagnosed with SCD) reduces RBC sickling via a reduction in levels of 2,3-diphosphoglycerate (2,3-DPG), which in turn reduces the polymerization of sickle Hgb (HgbS) into rigid aggregates that deform the cell. Furthermore, in some embodiments, PKR activation may contribute to overall RBC membrane integrity via increasing levels of adenosine triphosphate (ATP), which is predicted to reduce vaso-occlusive and hemolytic events which cause acute pain crises and anemia in SCD patients.

A PKR Activating Compound, such as Compound 1, is useful to promote activity in the glycolytic pathway. As the rate-limiting enzyme that catalyzes the last step of glycolysis, PKR directly impacts the metabolic health and primary functions of RBCs. PKR Activating Compounds (e.g., Compound 1), are useful to decrease 2,3-DPG and increase ATP. PKR Activating Compounds (e.g., Compound 1) are also useful to increase Hgb oxygen affinity in RBC. The disclosure is based in part on the discovery that PKR activation is a therapeutic modality for SCD, whereby HgbS polymerization and RBC sickling and hemolysis are reduced via decreased 2,3-DPG and increased ATP levels.

One aspect of this disclosure is targeting PKR activation to reduce 2,3-DPG levels, based on PKR's role in controlling the rate of glycolysis in RBCs. Increased activity of PKR tends to deplete organic phosphate precursors upstream of phosphoenolpyruvate, including 2,3-DPG. A decrease in 2,3-DPG with PKR activation has been demonstrated in preclinical studies and in healthy volunteers (e.g., FIGS. 18A, 18B, 19, 22, 30, 35, 36, 37, 43, 44, 45, 48, and 50 ). Additionally, PKR activation has been observed to increase ATP in these same studies (e.g., FIGS. 20A, 20B, 21, 40, 41, and 48 )) 3. NADH, generated along with ATP during glycolysis, is essential to reduce methemoglobin to Hb, thus reducing potential for oxidative stress. Furthermore, ATP plays a role in maintaining lipid asymmetry and ion gradients across the RBC membrane. Accordingly, elevating ATP levels is likely to have broad beneficial effects. Therefore, activation of PKR offers the potential for a 2,3-DPG effect (i.e., reduced cell membrane damage from HgbS polymerization) that is augmented by ATP support for membrane integrity. It is via these changes that a PKR activator is could positively impact physiological changes that lead to the clinical pathologies of SCD (FIG. 3 ). In another aspect, the disclosure relates to a method of improving the anemia and the complications associated with anemia in SCD patients (e.g., ≥12 years of age) with Hgb SS or Hgb SB⁰-thalassemia.

As illustrated in FIG. 4 , RBC metabolism utilizes glycolysis in order to generate ATP. 2,3-DPG is an intermediate in the glycolytic pathway and accumulates in RBCs under certain physiologic conditions. 2,3-DPG plays an important role in the ability of hemoglobin to bind oxygen. 2,3-DPG selectively binds to deoxyhemoglobin, making it harder for oxygen to bind hemoglobin and more likely to be released to adjacent tissues. 2,3-DPG is part of a feedback loop that can help prevent tissue hypoxia in conditions where it is most likely to occur. Under conditions of low tissue oxygen concentration such as high altitude, airway obstruction, or congestive heart failure, RBCs activate the Lubering-Rappoport shunt, a branch of the glycolytic pathway, to generate more 2,3-DPG. The accumulation of 2,3-DPG decreases the affinity of hemoglobin for oxygen eventually releasing it into the tissues that need it most.

PKR activation has potential to reduce both hemoglobin sickling and hemolysis via a reduction in 2,3-DPG and an increase in ATP. PKR activation depletes 2,3-DPG and increases ATP levels, thus increasing the energy supply of cells. Increasing cellular ATP may enhance the RBCs' ability to repair membrane damage and tolerate deformation in capillaries. Combining these two activities, a PKR activator has the potential to reduce the likelihood of sickling and increase the ability of RBCs to transit through small blood vessels without hemolysis. As illustrated in FIG. 4 , the multimodal action of a PKR-agonist (e.g., Compound 1) may increase hemoglobin levels and reduce VOCs in SCD patients. The studies described in the Examples demonstrate the Compound 1 mechanism of action.

Compound 1 Increases Hemoglobin Oxygen Affinity

Applicants have discovered that the compound (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one (“Compound 1”) or a pharmaceutically acceptable salt thereof, increases oxygen affinity of hemoglobin as measured by a left shift in the partial pressure of oxygen at 50% hemoglobin saturation (p50). Reduction in p50 indicates an increase in hemoglobin affinity for oxygen.

Applicants have discovered a method of increasing the oxygen affinity of hemoglobin A (HgbA) in red blood cells (RBCs). A method of treatment, can comprise administering to a patient (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one or a pharmaceutically acceptable salt thereof, in an amount effective to increase oxygen affinity of HbA. A method of treatment, can comprise administering to a patient (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one or a pharmaceutically acceptable salt thereof, in an amount effective to increase oxygen affinity of HgbA.

Applicants have discovered a method of increasing the oxygen affinity of hemoglobin A (HgbA) in red blood cells (RBCs). In human clinical studies, Compound 1 exhibited dose linear and time-independent PK, and the PD activity was observed at all dose levels after 24 h (decreased 2,3-DPG, p<0.0001) and after 14-days (increased ATP, p<0.0001) of dosing. Healthy volunteers who received Compound 1 experienced a decrease in p50p50 relative to baseline and relative to healthy volunteers who received placebo, reflecting an increase in oxygen affinity, while subjects who received the placebo did not. The biologic consequence of this PD response was an increase in oxygen affinity (decreased p50, p<0.0001) within 24 h of Compound 1 dosing and a decrease in absolute reticulocyte counts (p<0.0001) with a slight increase in hemoglobin levels (ns) by Day 4 of the dosing period in all Compound 1 dose cohorts. The increase in hemoglobin A (HgbA) affinity for oxygen in healthy subjects can be seen by the oxyhemoglobin dissociation curve (p50; partial pressure of O2 at which 50% of hemoglobin is saturated with O2) after a single dose and after 14-day dosing of Compound 1. A mean decrease in 2,3-DPG and p50, and a mean increase in ATP, relative to baseline, was observed in both the single ascending dose (SAD) and multiple ascending dose (MAD) cohorts. Within 24 hr of a single dose of Compound 1, a decrease in 2,3-DPG with a corresponding increase in p50 was observed. Healthy volunteers (having normal hemoglobin, or HgbA) who received Compound 1 experienced a change (decrease) in p50 relative to baseline, while subjects who received the placebo did not. In the SAD cohorts, the subjects' p50 (PO2 at 50% hemoglobin saturation) were determined 24-hours post-dose. The pp50 values measured 24 hours after a single dose of Compound 1 were reduced at all dose levels tested (median reduction ranged from ˜3-5 mmHg). In the MAD cohorts, the subjects' p50 (PO2 at 50% hemoglobin saturation) were determined on day 14. p50 values measured after 14 days of once or twice daily dosing were reduced at all dose levels tested (median reduction ranged from ˜3-5 mmHg).

In some embodiments, a method of treatment comprises administering Compound 1 to a patient in an amount effective to increase the oxygen affinity of RBC from the patient (e.g., as measured by a reduction in p50 from a blood sample take 24 hours after administration of Compound 1 to the patient). In some embodiments, a method of treatment can comprise administering Compound 1 to a patient in an amount effective to reduce the p50p50 (pO2 at 50% hemoglobin saturation) measured 24 hours after administration of Compound 1 relative to baseline by more than 0.2 mmHg (mean absolute change), including reducing the effective p50 of a patient by 1, 2, 3, 4, 5, or more mmHg (including reductions of about 2.9, 3.4, 4.9 and 5.1 mmHg) relative to baseline at 24 hours after administration of Compound 1. In some embodiments, a method of treatment comprises administering Compound 1 followed by measuring a decrease in p50 relative to baseline in the patient (e.g., from a blood sample) 24 hours after the administration of Compound 1, reflecting an increase in oxygen affinity. In some embodiments, due to the lack of cytochrome P450 induction and the extended half-life of the pharmacodynamic effect, the compound is taken on a QD regimen.

A method of treating a patient diagnosed with a hemoglobinopathy, can comprise administering Compound 1 (or a pharmaceutically acceptable salt thereof) in an amount effective to increase oxygen affinity of HbS in the patient or to provide a left shift in the point of sickling (PoS) with an increase in the EImin in the patient, or a combination thereof. For example, the hemoglobinopathy can be Sickle Cell Disease. In another embodiment, a method of treating a patient diagnosed with a hemoglobinopathy can comprise administering Compound 1 (or a pharmaceutically acceptable salt thereof) in an amount effective to increase intracellular ATP levels in the RBC or to improve the membrane function, for example in Sickle Cell Disease or beta-thalassemia.

A method of treatment, can comprise administering to a patient (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one or a pharmaceutically acceptable salt thereof, in an amount effective to increase oxygen affinity of HbS. A method for increasing oxygen affinity of sickle hemoglobin (HbS) in vivo in a patient in need thereof can comprise administering to said patient a sufficient amount of (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one or a pharmaceutically acceptable salt thereof. In some embodiments, the administration of a single dose of (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one or a salt thereof can increase the oxygen affinity of said HbS in the patient.

A method for increasing oxygen affinity of sickle hemoglobin (HbS) in vivo in a patient in need thereof can comprise administering to said patient a sufficient amount of (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one or a pharmaceutically acceptable salt thereof, to increase oxygen affinity of the blood of a SCD patient. In some embodiments, the administration of a single dose of (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one or a salt thereof can increase the oxygen affinity of said HbS in the patient.

In some embodiments, methods of increasing the oxygen affinity of hemoglobin in red blood cells (RBCs) can comprise contacting the RBCs with an amount of Compound 1 under conditions and for a time effective to reduce the amount of 2,3-DPG in the RBCs.

In some embodiments, methods of treatment comprise administering a pharmaceutical composition comprising Compound 1 to a patient diagnosed with a hemolytic anemia in an amount effective to increase hemoglobin oxygen affinity in RBC, including a patient diagnosed with Sickle Cell Disease.

Compound 1 Increases ATP and Reduces 2,3-DPG Concentrations in Blood

Another aspect of the disclosure is the recognition that activation of PKR can reduce 2,3-diphosphoglycerate (2,3-DPG), which inhibits Hgb deoxygenation/increases oxygen affinity of HgbS and leads to decreased deoxy-HgbS (and, therefore, sickling), as well as can increase ATP, which promotes membrane health and reduces hemolysis. Accordingly, in one embodiment, PKR activation (e.g., by administration of a therapeutically effective amount of Compound 1 or a pharmaceutically acceptable salt thereof to a patient diagnosed with SCD) reduces RBC sickling via a reduction in levels of 2,3-diphosphoglycerate (2,3-DPG), which in turn reduces the polymerization of sickle Hgb (HgbS) into rigid aggregates that deform the cell. Furthermore, in some embodiments, PKR activation may contribute to overall RBC membrane integrity via increasing levels of adenosine triphosphate (ATP), which is predicted to reduce vaso-occlusive and hemolytic events which cause acute pain crises and anemia in SCD patients.

In some embodiments, Compound 1 is administered in a dose that is pharmacodynamically effective. In some embodiments, Compound 1 is administered in a dose resulting in a reduction in RBC 2,3-DPG in the patient (e.g., measured in the blood of the patient 6 hours after administration of Compound 1). The reduction of 2,3-DPG can be measured in patient blood by a qualified LC-MS/MS method for the quantitation of 2,3-DPG in blood, or using a commercially available kit. In some embodiments, a method of treatment can comprise administering Compound 1 to a patient in an amount effective to reduce 2,3-DPG levels by one or more of the following after administering a dose of Compound 1, relative to patient baseline:

-   -   at least 10% after 6 hours (e.g., by more than 7.8% after 6         hours, by at least 18% after 6 hours, or by about 18-29% after 6         hours),     -   by at least 10% after 8 hours (e.g., by more than 7.6% after 8         hours, by at least 17% after 8 hours, or by about 17-29% after 8         hours),     -   by at least 10% after 12 hours (e.g., by more than 4.0% after 12         hours, by at least 25% after 12 hours, or by about 25-44% after         8 hours),     -   by at least 10% after 16 hours (e.g., by more than 6.0% after 16         hours, by at least 33% after 16 hours, or by about 33-50% after         16 hours),     -   by at least 10% after 24 hours (e.g., by more than 2.0% after 24         hours, by at least 31% after 24 hours, or by about 31-49% after         24 hours),     -   by at least 10% after 36 hours (e.g., by more than 6.9% after 36         hours, by at least 33% after 36 hours, or by about 33-47% after         36 hours),     -   by at least 10% after 48 hours (e.g., by more than 15% after 48         hours, by at least 29% after 48 hours, or by about 29-48% after         48 hours), and     -   by at least 10% after 72 hours (e.g., by more than 6.9% after 72         hours, by at least 18% after 72 hours, or by about 18-33% after         72 hours).

In some embodiments, Compound 1 is administered in a dose resulting in an increase in RBC ATP in the patient (e.g., measured in the blood of the patient 6 hours after administration of Compound 1). In some embodiments, a method of treatment comprises administering Compound 1 to a patient in an amount effective to elevate ATP levels in the patient, relative to baseline, for one or more consecutive days (e.g., 1-14 days or more), wherein the levels of ATP remain elevated in the patient ATP levels remain elevated, relative to baseline, for 60 hours after the last dose of Compound 1. ATP is measured in RBCs. For example, in some embodiments, a method of treatment comprises administering Compound 1 daily to a patient for 14 consecutive days in an amount to increase ATP levels in the patient by one or more of the following amounts, relative to patient baseline:

-   -   more than 0% within less than 6 hours after administration of         Compound 1 on day 14 (e.g., by at least 41% within 6 hours, or         by about 41-55% within 6 hours),     -   more than 2.8% after 6 hours after administration of Compound 1         on day 14 (e.g., by at least 44% after 6 hours, or by about         44-48% after 6 hours),     -   more than 0% after 8 hours after administration of Compound 1 on         day 14 (e.g., by at least 47% after 12 hours, or by about 47-58%         after 8 hours),     -   more than 2.3% after 12 hours after administration of Compound 1         on day 14 (e.g., by at least 45% after 12 hours, or by about         45-56% after 12 hours),     -   more than 0% after 16 hours after administration of Compound 1         on day 14 (e.g., by at least 44% after 16 hours, or by about         44-57% after 16 hours),     -   more than 2.9% after 24 hours after administration of Compound 1         on day 14 (e.g., by at least 55% after 24 hours, or by about         55-64% after 24 hours),     -   more than 4.7% after 48 hours (e.g., by at least 52% after 48         hours, or by about 52-59% after 48 hours), and     -   more than 2.2% after 72 hours after administration of Compound 1         on day 14 (e.g., by at least 49% after 72 hours, or by about         49-54% after 72 hours).

In some embodiments, a method of treatment can comprise administering Compound 1 to a patient for multiple consecutive days (e.g., 14 days or more) in an amount and dose interval effective to reduce 2,3-DPG levels, relative to baseline, of at least about 25% when tested 24 hours after administration of the first dose on day 1 and at least about 40% when tested 24 hours after administration of the first dose on day 14. For example, in some embodiments, a method of treatment comprises administering Compound 1 daily to a patient for 14 consecutive days in an amount to reduce 2,3-DPG levels by one or more of the following amounts, relative to patient baseline:

-   -   more than 7.6% within less than 6 hours after administration of         Compound 1 on day 14 (e.g., by at least 42% within 6 hours, or         by about 42-59% within 6 hours),     -   more than 10.9% after 6 hours after administration of Compound 1         on day 14 (e.g., by at least 44% after 6 hours, or by about         44-53% after 6 hours),     -   more than 1.6% after 8 hours after administration of Compound 1         on day 14 (e.g., by at least 44% after 12 hours, or by about         44-54% after 8 hours),     -   more than 1.6% after 12 hours after administration of Compound 1         on day 14 (e.g., by at least 42% after 12 hours, or by about         42-55% after 12 hours),     -   more than 5.3% after 16 hours after administration of Compound 1         on day 14 (e.g., by at least 42% after 16 hours, or by about         42-52% after 16 hours),     -   more than 10.7% after 24 hours after administration of Compound         1 on day 14 (e.g., by at least 44% after 24 hours, or by about         44-52% after 24 hours),     -   more than 1% after 48 hours (e.g., by at least 34% after 48         hours, or by about 34-44% after 48 hours), and     -   more than 7% after 72 hours after administration of Compound 1         on day 14 (e.g., by at least 20% after 72 hours, or by about         20-32% after 72 hours).

Compound 1 Reduces Sickling in SCD Patient RBCs

Compound 1 can improve RBC membrane integrity. One aspect of the disclosure is the recognition that activation of PKR can reduce 2,3-diphosphoglycerate (2,3-DPG), which leads to decreased deoxy-HgbS (and, therefore, sickling), as well as can increase ATP, which promotes membrane health and reduces hemolysis.

In some embodiments, the disclosure relates to a method of improving RBC membrane function in a patient diagnosed with sickle cell disease (SCD), comprising administering to the patient a sufficient amount of (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one or a pharmaceutically acceptable salt thereof. In some embodiments, improving RBC membrane function comprises improving RBC membrane response to an osmotic gradient, as evidenced by a shift toward normal in Omin and Ohyper.

A method for inhibiting sickling of HbS in a patient diagnosed with Sickle Cell Disease, (SCD), can comprise administering to said patient a sufficient amount of a composition comprising (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one or a pharmaceutically acceptable salt thereof. A method of treating a patient diagnosed with Sickle Cell Disease (SCD), can comprise administering to said patient a therapeutically effective single dose of (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one or a pharmaceutically acceptable salt thereof, such that the patient experiences a left shift in the point of sickling (PoS) with an increase in the EImin after 24 hours. A method of treatment, can comprise administering to a patient (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one or a pharmaceutically acceptable salt thereof, in an amount effective to result in a left shift in the point of sickling (PoS) with an increase in the EImin in the patient.

A method for inhibiting sickling of HbS in a patient diagnosed with Sickle Cell Disease, (SCD), can comprise administering to said patient a sufficient amount of a composition comprising (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one or a pharmaceutically acceptable salt thereof.

A method of treating a patient diagnosed with Sickle Cell Disease (SCD), can comprise administering to said patient a therapeutically effective single dose of (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one or a pharmaceutically acceptable salt thereof, such that the patient experiences a left shift in the point of sickling (PoS) with an increase in the EImin after 24 hours.

In some embodiments, the disclosure relates to a method of reducing RBC turnover in a patient diagnosed with sickle cell disease (SCD), comprising administering to the patient a sufficient amount of a PKR Activating Compound, e.g., (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one or a pharmaceutically acceptable salt thereof.

Treating Pediatric Patients with Compound 1

In some embodiments, methods of treating sickle cell disease or other hemoglobinopathy comprise administering Compound 1 once per day (QD) to adults and pediatric patients 12 years of age and older. In some embodiments, methods of treating sickle cell disease or other hemoglobinopathy comprise administering Compound 1 once per day (QD) to adults and pediatric patients younger than 12 years of age. In some embodiments, methods of treating sickle cell disease or other hemoglobinopathy comprise administering Compound 1 once per day (QD) to pediatric patients 2-12 years of age. In some embodiments, methods of treating sickle cell disease or other hemoglobinopathy comprise administering Compound 1 once per day (QD) to adults and pediatric patients up to age 2 years of age.

Compound 1 has the potential to be a foundational treatment for patients early in life. Patients may benefit from being treated early to potentially lessen the impact of the disease. For example, as further described in Example 12, Compound 1 has not shown evidence of aromatase inhibition, CYP induction or CYP inhibition.

Compound 1 is well-tolerated and has not shown evidence of inhibition of aromatase, an enzyme involved in converting testosterone to estrogen, which may permit dosing in a broad range of patients, including both pediatric and adult populations (e.g., treatment of patients ages 12 and older diagnosed with SCD or other conditions, or treatment of pediatric patients younger than 12 diagnosed with SCD), as it does not lead to alterations in the hormones that affect pediatric growth and development. Aromatase is an enzyme encoded by the CYP19A1 gene. It is located in the endoplasmic reticulum of estrogen-producing cells and catalyzes the rate-limiting step in the conversion of androgens to estrogens in many tissues. Aromatase is a cytochrome P-450 hemoprotein-containing enzyme complex that catalyzes the rate-limiting step in the production of estrogens, i.e. the conversion of androstenedione and testosterone, via three hydroxylation steps, to estrone and estradiol. Aromatase activity is present in many tissues, such as the ovaries, adipose tissue, muscle, liver, breast tissue, and in malignant breast tumors. The main sources of circulating estrogens are the ovaries in premenopausal women and adipose tissue in post-menopausal women. Aromatase catalyzes the conversion of androgens to estrone (E1), which is further converted to the potent estrogen estradiol (E2) by the enzyme 17β-HSD type 1 in the granulosa cell.

Aromatase is a key enzyme in the steroidogenic pathway that catalyzes the conversion of androgens, including testosterone, into estradiol. Inhibition of aromatase increases testosterone and decreases estradiol, both important hormones for human sexual development during childhood. Sickle cell disease is an inherited disorder manifesting as early as 6 months old. Activators of PKR, including Compound 1, are promising investigational therapies being developed for the treatment of Sickle Cell Disease. Aromatase inhibition has been observed with AG-348 (mitapivat) a clinical PKR activator (Yang et al. 2018; Grace et al. 2019). Absence of aromatase inhibition is a desired property for therapies intended to treat children and adolescents, including those with sickle cell disease. Affecting the production of these sex hormones in children and adolescents could have adverse effects on a child/adolescent's sexual maturation/development and growth. Based on the preclinical studies and confirmed by the healthy volunteers receiving Compound 1 continuously for up to 14 days, Compound 1 has no effect on estradiol and testosterone levels.

Once-Daily (OD) Dosing of Compound 1

Compound 1 demonstrates pharmacological response in healthy volunteers dosed with a single daily dose of 400 mg that is not directly related to plasma concentrations. Maximal decrease in blood levels of the target engagement biomarker 2,3-DPG occurs ˜16 to 24 h post-dose, long after the plasma Cmax, and is sustained up to ˜48 h post dose (e.g, FIG. 43 ). Furthermore, after 14 days of dosing, the downstream effect on hemoglobin oxygen affinity is similar with once daily doses of 400 mg or twice daily dosing of 200 mg (e.g., FIG. 42 ).

In healthy volunteers receiving a single dose of Compound 1, dose normalized Cmax and AUC increased with increasing doses ≥700 mg suggesting greater than dose proportional increases in exposure at the highest doses tested (FIG. 31A). In healthy volunteers receiving multiple doses of Compound 1, a dose linear exposure was observed across all dose levels tested and PK parameters (Cmax and AUC) remained constant on Day 14 compared to Day 1 indicating Compound 1 demonstrates time-independent pharmacokinetics (FIG. 31B). After multiple-doses (every 12 or 24 hours for 14 consecutive days), dose linear exposure was observed across all dose levels tested and PK parameters (Cmax and AUC) remained similar on Day 14 compared to Day 1, indicating time-independent PK. The underlying properties of Compound 1 driving the observed time-independent PK include a lack of observed CYP inhibition or induction demonstrated by Compound 1 in vitro, thereby reducing the risk of inhibiting or inducing its own clearance as well as reducing the risk for drug-drug interactions.

Underlying the observed constant exposure over time is the lack of CYP inhibition or induction demonstrated by Compound 1 in vitro, thereby reducing risk of inhibiting or inducing its own metabolism as well as reducing the risk for drug-drug interactions due to CYP's effects on pharmacokinetics of other drugs through changes in plasma concentration. SCD patients typically take numerous concurrent medications to address their disease. The body will naturally break down many of these medications through CYP. When the expression of these enzymes is inhibited or induced by another medication, it can impact the efficacy of concurrent medications. Limiting the potential for drug-drug interactions is imperative to effectively treat this patient population. Compound 1 has been observed preclinically to have no significant impact on CYP enzyme inhibition or induction. Some compounds according to the present invention, including the physiologically acceptable salts, exhibit favourable, that is low Cytochrome P450 (CYP) induction potential. CYP induction can affect the pharmacokinetics of a drug molecule upon multiple dosing, which can result in pharmacokinetic drug-drug interactions with coadministered drugs (e.g., by increasing the metabolic clearance of co-administered CYP3A4 substrates), or can cause loss of drug exposure due to autoinduction. CYP induction can lead to decreased exposure of the inducing drug (e.g. autoinduction) or decreased exposure of a coadministered drug metabolized by the induced enzyme. CYP induction can also lead to an increase in the metabolism of a drug causing changes in pharmacological (active metabolite) and toxicological (toxic metabolite) outcomes. Characterizing the induction potential of discovery or development drug candidates has become an important screen throughout the pharmaceutical industry. A PXR transactivation assay is used to assess the induction potential of CYP3A4. Reduced inhibition of CYP isozymes may translate into a reduced risk for undesirable drug-drug interactions which is the interference of one drug with the normal metabolic or pharmacokinetic behavior of a co-administered drug.

Compound 1 has not demonstrated any preclinical evidence of arrhythmia risk, mutagenicity, or nonspecific binding activity for panels of receptors, enzymes, ion channels, and kinases in vitro, suggesting a potentially positive tolerability profile.

Treating Hemoglobinopathies with Compound 1

Hemoglobinopathies are a diverse range of rare inherited genetic disorders in which there is production of an abnormal hemoglobin, dysregulation of the amount of hemoglobin, or the complete absence of one of the hemoglobin subunits. Compound 1's mechanism of action supports its use across a number of adjacent indications. Compound 1 is a potent activator of PKR, designed to improve RBC metabolism, function and survival, by impacting the critical glycolytic pathway. An increase in ATP resulting from the activation of PKR may improve RBC membrane health and integrity. Applicant believes this approach will improve hemoglobin-related diseases through increased RBC survival, reduce the hemolysis associated with beta thalassemia and alleviate the primary symptoms in patients.

One aspect of the disclosure relates to methods of treating a patient comprising the administration of a therapeutically effective amount of a pyruvate kinase R (PKR) activator to a patient in need thereof. Preferably, a patient diagnosed with a hemoglobinopathy is treated with a compound that is a PKR Activating Compound. The PKR activator can be a compound identified as a PKR Activating Compound or a composition identified as a PKR Activating Composition having an AC₅₀ value of less than 1 μM using the Luminescence Assay described in Example 2, or a pharmaceutically acceptable salt and/or other solid form thereof. One aspect of the disclosure relates to methods of treating a patient, such as a patient diagnosed with a hemoglobinopathy, comprising the administration of a therapeutically effective amount of Compound 1 or a pharmaceutically acceptable salt thereof. Methods of treating various hemoglobinopathy conditions can comprise the administration of a therapeutically effective amount of a PKR Activating Compound to a patient in need thereof. Various additional methods of administering a PKR Activating Compound to a patient diagnosed with a hemoglobinopathy are provided herein.

As used herein, the term “hemoglobinopathy” means any defect in the structure, function or expression of any hemoglobin of an individual, and includes defects in the primary, secondary, tertiary or quaternary structure of hemoglobin caused by any mutation, such as deletion mutations or substitution mutations in the coding regions of the β-globin gene, or mutations in, or deletions of, the promoters or enhancers of such genes that cause a reduction in the amount of hemoglobin produced as compared to a normal or standard condition. The term “hemoglobinopathy” further includes any decrease in the amount or effectiveness of hemoglobin, whether normal or abnormal, caused by external factors such as disease, chemotherapy, toxins, poisons, or the like, β-hemoglobinopathies contemplated herein include, but are not limited to, sickle cell disease (SCD, also referred to a sickle cell anemia or SCA), sickle cell trait, hemoglobin C disease, hemoglobin C trait, hemoglobin S/C disease, hemoglobin D disease, hemoglobin E disease, thalassemias, hemoglobins with increased oxygen affinity, hemoglobins with decreased oxygen affinity, unstable hemoglobin disease and methemoglobinemia.

In some embodiments, the hemoglobinopathy is a condition that can be therapeutically treated by PKR activation resulting from the administration of a therapeutically effective amount of Compound 1. Enhancement of PKR activity may also increase NADH levels and therefore ability to reduce methemoglobin to hemoglobin. The enzyme methemoglobin reductase utilizes NADH, which like ATP, is generated during glycolysis.

In some embodiments, the disease or disorder is selected from the group consisting of PKD, SCD, sickle cell anemia, thalassemia (e.g., beta-thalassemia or alpha-thalassemia), hereditary non-spherocytic hemolytic anemia, hemolytic anemia (e.g., chronic hemolytic anemia caused by phosphoglycerate kinase deficiency (PKD)), hereditary spherocytosis, hereditary elliptocytosis, abetalipoproteinemia (or Bassen-Kornzweig syndrome), paroxysmal nocturnal hemoglobinuria, acquired hemolytic anemia (e.g., congenital anemias (e.g., enzymopathies)), or anemia of chronic diseases.

In some embodiments, the method comprises administering a therapeutically effective amount of a Compound 1 for the treatment of a patient diagnosed with a condition selected from the group consisting of: hereditary non-spherocytic hemolytic anemia, hemolytic anemia (e.g., chronic hemolytic anemia caused by phosphoglycerate kinase deficiency), hereditary spherocytosis, hereditary elliptocytosis, abetalipoproteinemia (or Bassen-Kornzweig syndrome), paroxysmal nocturnal hemoglobinuria, acquired hemolytic anemia (e.g., congenital anemias (e.g., enzymopathies)), and anemia of chronic diseases. In some embodiments, the disease or disorder is hereditary non-spherocytic hemolytic anemia. In some embodiments, the disease or disorder is SCD (e.g., sickle cell anemia) or thalassemia (e.g., beta-thalassemia). In some embodiments, the disease or disorder is hemolytic anemia (e.g., in a patient diagnosed with PKD). In some embodiments, the disease or disorder is beta thalassemia. In some embodiments, the disease or disorder is SCD. In some embodiments, the disease or disorder is selected from the group consisting of SCD, sickle cell anemia, thalassemia (e.g., beta-thalassemia), hereditary non-spherocytic hemolytic anemia, hemolytic anemia (e.g., chronic hemolytic anemia caused by phosphoglycerate kinase deficiency), hereditary spherocytosis, hereditary elliptocytosis, abetalipoproteinemia (or Bassen-Kornzweig syndrome), paroxysmal nocturnal hemoglobinuria, acquired hemolytic anemia (e.g., congenital anemias (e.g., enzymopathies)), and anemia of chronic diseases.

In another embodiment, the present disclosure relates to a compound of Formula (I) or a pharmaceutical composition comprising a compound of the present disclosure and a pharmaceutically acceptable carrier used for the treatment of SCD, sickle cell anemia, thalassemia (e.g., beta-thalassemia), hereditary non-spherocytic hemolytic anemia, hemolytic anemia (e.g., chronic hemolytic anemia caused by phosphoglycerate kinase deficiency), hereditary spherocytosis, hereditary elliptocytosis, abetalipoproteinemia (or Bassen-Kornzweig syndrome), paroxysmal nocturnal hemoglobinuria, acquired hemolytic anemia (e.g., congenital anemias (e.g., enzymopathies)), or anemia of chronic diseases.

A method of treating a patient diagnosed with a hemoglobinopathy, can comprise administering a PKR Activating Compound in an amount effective to increase oxygen affinity of HbS in the patient or to provide a left shift in the point of sickling (PoS) with an increase in the deformability (EImin) in the patient, or a combination thereof. For example, the hemoglobinopathy can be Sickle Cell Disease or beta-thalassemia. In some embodiments, a patient diagnosed with a hemoglobinopathy is treated with Compound 1 or a pharmaceutically acceptable salt thereof. In some embodiments, the patient is diagnosed with Sickle Cell Disease or beta-thalassemia.

Patient Hemoglobin Genotype

Compound 1 can be administered to subjects having various genotypes. In some embodiments, Compound 1 can be administered to red blood cells of a subject having normal hemoglobin (e.g., HbA, HbA1, HbA2, HbE, HbF, HbS, HbC, HbH, and HbM, and HbF <2% of total hemoglobin). In some embodiments, methods of treatment comprise the step of administering a pharmaceutical composition to a patient diagnosed with hemoglobinopathies comprising hemoglobin genotypes other than HbA. In some embodiments, the patient is diagnosed with a condition previously confirmed by hemoglobin electrophoresis or genotyping. In some embodiments, the patient can be diagnosed with a genotype indicating one of the following hemoglobin genotypes: Hgb SS, Hgb Sβ+-thalassemia, Hgb Sβ0-thalassemia, or Hgb SC, which is often determined as part of universal newborn screening available in the majority of U.S. states. In some embodiments, the disclosure relates to a method of improving the anemia and the complications associated with anemia in SCD patients (e.g., ≥12 years of age, and/or <12 years of age) with Hgb SS or Hgb SB0-thalassemia. In some embodiments, Compound 1 is administered to a patient diagnosed with a SCD genotype comprising HbS. In some embodiments, methods of treatment can comprise administering compound 1 to a patient diagnosed with a HbSS disease or sickle cell anemia (i.e., homozygote for the S globin), HbS/b-0 thalassemia (double heterozygote for HbS and b-0 thalassemia), HbS/b+ thalassemia, HbSC disease (i.e., double heterozygote for HbS and HbC), HbS/hereditary persistence of fetal Hb (S/HPHP), HbS/HbE syndrome, or rare combinations of HbS (e.g., HbD Los Angeles, G-Philadelphia, or HbO Arab).

Treating Sickle Cell Disease (SCD) with Compound 1

In some embodiments, methods of treatment comprise the step of administering Compound 1 to a patient diagnosed with SCD, where the patient is further characterized by one or more of the following: (1) previously confirmed hemoglobin genotype selected from the group consisting of Hb SS and Hb SC, (2) age 12 to 65 years, (3) patients having had ≤6 vaso-occlusive crises (VOCs) within the past 12 months prior to receiving Compound 1, (4) no PRBC transfusion within 30 days of first receiving Compound 1; and, optionally, (5) concomitant hydroxyurea use.

Referring to the schematic in FIG. 5 , SCD arises from abnormalities in the beta subunit, specifically when a genetic mutation creates the variant form of the beta subunit, called ßs. SCD is an autosomal recessive disorder characterized by a point mutation in the beta-globin gene that results in a single amino acid substitution that predisposes polymerization of deoxy hemoglobin. This polymerization results in deformation of RBCs into a less-pliable, sickle shape. The sickle-shaped RBCs also exhibit membrane damage in the form of altered surface lipids and are prone to adhere to vascular endothelium and white blood cells in small blood vessels in peripheral tissues that can block blood flow to organs and cause acute and painful VOC events. As a result of this obstruction, there is destruction of some RBCs, or hemolysis. This destruction of RBCs leads to the intravascular release of hemoglobin which itself can generate highly damaging oxidative chemicals. The release of hemoglobin and other cytoplasmic molecules from RBCs also trigger signaling cascades that lead to platelet activation, increased endothelial adhesion, inflammation in the vasculature and further obstruction of blood vessels. Acute complications of VOC cause tissue damage due to the lack of oxygen delivery to tissues, resulting in severe pain and symptoms, such as acute chest syndrome. Tissues that are deprived of oxygen are subject to ischemia and reperfusion injuries that can cause damage and long-term organ failure.

Sickle cell disease (SCD) is a chronic hemolytic anemia caused by inheritance of a mutated form of hemoglobin (Hgb), sickle Hgb (HgbS). It is the most common inherited hemolytic anemia, affecting 70,000 to 80,000 patients in the United States (US). SCD is characterized by polymerization of Hgb S in red blood cells (RBCs) when HgbS is in the deoxygenated state (deoxy-HgbS), resulting in a sickle-shaped deformation. Sickled cells aggregate in capillaries precipitating vaso-occlusive events that generally present as acute and painful crises resulting in tissue ischemia, infarction, and long-term tissue damage. RBCs in patients with SCD tend to be fragile due to repeated cycles of sickling and mechanical deformation, which induce damage including membrane dysfunction. Reactive oxygen species caused by HbS lead to oxidative damage. Together, these sources of damage lead tohemolysis and chronic anemia. Finally, damaged RBCs have abnormal surfaces that adhere to and damage vascular endothelium, provoking a proliferative/inflammatory response that underlies large-vessel stroke and potentially pulmonary-artery hypertension. Collectively, these contribute to the significant morbidity and increased mortality associated with this disease.

The described clinical symptoms of SCD are largely due to perturbations in RBC membrane shape and function resulting from aggregation of HgbS molecules. Unlike normal Hgb, HgbS polymerizes when it is in the deoxygenated state and ultimately causes a deformed, rigid cell that is unable to pass through small blood vessels, thereby blocking normal blood flow through microvasculature. The loss of membrane elasticity also increases hemolysis and clearance by the spleen, reducing RBC longevity. Furthermore, decreased cellular ATP and oxidative damage contribute to a sickle RBC membrane that is stiffer and weaker than that of normal RBCs. The damaged membrane has a greater propensity for adhering to vasculature, leading to hemolysis, increased aggregation of sickled RBCs, and increased coagulation and inflammation associated with vaso-occlusive crises.

The underlying cause of sickling is the formation of rigid deoxy-HgbS aggregates that alter the cell shape and consequently impact cellular physiology and membrane elasticity. These aggregates are highly structured polymers of deoxygenated HgbS; the oxygenated form does not polymerize. Polymerization is promoted by a subtle shift in conformation from the oxygen-bound relaxed (R)-state to the unbound tense (T)-state that exposes the mutant hydrophobic valine residue at position 6 of the β-globin chain. These valine residues within the β-chain of HgbS are able to interact in a specific and repetitive manner, facilitating polymerization.

The concentration of deoxy-HgbS depends on several factors, but the predominant factor is the partial pressure of oxygen (PO₂). Oxygen reversibly binds to the heme portions of the Hgb molecule. As oxygenated blood flows via capillaries to peripheral tissues and organs that are actively consuming oxygen, PO₂ drops and Hgb releases oxygen. The binding of oxygen to Hgb is cooperative and the relationship to PO₂ levels fits a sigmoidal curve (FIG. 6 ). This relationship can be impacted by temperature, pH, carbon dioxide, and the glycolytic intermediate 2,3-DPG. 2,3-DPG binds within the central cavity of the Hgb tetramer, causes allosteric changes, and reduces Hgb's affinity for oxygen. 2,3-DPG is normally increased in response to anemia, and is therefore higher in SCD patients. Therapeutic approaches that increase oxygen affinity (i.e., reduce deoxygenation) of HgbS will decrease the rate of polymer formation, changes to the cell membrane, and clinical consequences associated with certain hemoglobinopathy conditions such as SCD. These changes would be observed at the cellular level but also would be reflected in clinical measurements such as Hb, RBC and reticulocyte counts, as well as in measures of hemolysis such as LDH levels in plasma or serum.

SCD is the most common type of hemoglobinopathy, a diverse range of rare inherited genetic disorders that affect hemoglobin, the iron-containing protein in RBCs responsible for transporting oxygen in the blood. In SCD, a structural abnormality in hemoglobin results in RBCs with a sickle-shaped deformation after off-loading oxygen to tissues. These sickle RBCs can aggregate in tissue blood vessels and block blood flow and oxygen delivery to organs, which can lead to acute and painful VOC events that result in tissue ischemia, infarction, and long-term tissue damage. In addition, sickle RBCs tend to be fragile due to sickling and have a half-life of 10 to 20 days versus normal RBCs, which have a half-life of 90 to approximately 120 days. This fragility leads to hemolysis, or the destruction of sickle RBCs, and chronic anemia, or reduced levels of RBCs and total hemoglobin. Additionally, damaged RBCs release factors that are detrimental to the vascular endothelium and can induce an inflammatory response that underlies large-vessel stroke and pulmonary arterial hypertension. On average, adult SCD patients are hospitalized three times per year and have significant morbidity and increased mortality.

The VOC events generally begin early in childhood and may lead to heart and lung complications, renal dysfunction, priapism, spleen enlargement and failure, stroke, retinopathy and mental and physical disabilities. Chronic pain is common, occurring on approximately 55% of days, as self-reported in SCD patients. Acute chest syndrome occurs in approximately half of all patients with SCD and is a leading cause of hospitalization and death among patients with SCD. Stroke occurs in 11% of patients with SCD by the age of 20 and in 24% of patients by the age of 45. Approximately 10% of patients with SCD suffer from pulmonary hypertension. Some patients with SCD experience end-stage renal failure that requires dialysis and portends a one-year mortality of 26%. Adult patients with SCD are hospitalized 1.5 times per year on average, and one-third of patients with SCD are readmitted to the hospital within 30 days of initial hospitalization.

SCD clinically manifests with potentially severe pathological conditions associated with substantial physical, emotional, and economic burden. For instance, acute vaso-occlusive pain crises can be debilitating and necessitate rapid medical response. Chronic hemolytic anemia causes fatigue and often necessitates blood transfusions and supportive care. Over time, impaired oxygen transport through microvasculature precipitates organ and tissue damage. While there are a number of options available for treating symptoms, overall disease management would benefit from therapies that target upstream processes to prevent vaso-occlusion and hemolysis.

As provided herein, certain methods of treating SCD preferably include administration of a therapeutically effective amount of a PKR Activating Compound (e.g., Compound 1) that reduces HgbS polymerization, for example by increasing HgbS affinity for oxygen. Methods of treating SCD also preferably include administration of a therapeutically effective amount of a compound (e.g., Compound 1) that reduces HgbS polymerization, for example by increasing HgbS affinity for oxygen. Methods of lowering 2,3-DPG and/or increasing ATP levels in human RBCs comprise administering a PKR Activating Compound, such as Compound 1. Methods of lowering 2,3-DPG and/or increasing ATP levels in human RBCs also comprise administering a PKR Activating Compound, such as Compound 1. Together these effects are consistent with providing therapies to reduce HgbS sickling and to improve RBC membrane health, presenting a unique disease-modifying mechanism for treating SCD.

A PKR Activator Compound, such as Compound 1, can be administered orally, once-daily, for the treatment of SCD. SCD, one of the most common single-gene disorders in the world, is a chronic hemolytic anemia that affects hemoglobin, the iron-containing protein in red blood cells, or RBCs, that delivers oxygen to cells throughout the body. SCD is often characterized by low hemoglobin levels, painful vaso-occlusive crises, or VOCs, progressive multi-organ damage and early death. Compound 1 is a potent activator of pyruvate kinase-R, or PKR, designed to improve RBC metabolism, function and survival, and potentially resulting in both increased hemoglobin levels and reduced VOCs. Unlike other emerging SCD therapies, Compound 1 modulates RBC metabolism by impacting two critical pathways through PKR activation: a decrease in 2,3 diphosphoglycerate, or 2,3-DPG, which increases oxygen affinity and an increase in adenosine triphosphate, or ATP, which may improve RBC and membrane health and integrity. This multi-modal approach may improve hemoglobin levels through increased RBC survival and decrease VOCs through reduced RBC sickling. Compound 1 has the potential to become the foundational standard of care for SCD patients by modifying the disease at an early stage and potentially preventing end-organ damage, reducing hospitalizations, and improving the patients' overall health and quality of life.

In some embodiments, the disclosure relates to a method of increasing Hb concentration in a patient diagnosed with sickle cell disease (SCD), comprising orally administering to the patient in need thereof a therapeutically effective amount of (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one or a pharmaceutically acceptable salt thereof, once per day (QD). In some embodiments, the disclosure relates to a method of increasing Hb concentration in a patient diagnosed with sickle cell disease (SCD), comprising administering to the patient a sufficient amount of a PKR Activating Compound, e.g., (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one or a pharmaceutically acceptable salt thereof.

In some embodiments, the disclosure relates to a method of reducing point of sickling (POS) in a patient diagnosed with sickle cell disease (SCD), comprising administering to the patient a sufficient amount of a PKR Activating Compound, e.g., (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one or a pharmaceutically acceptable salt thereof.

In some embodiments, the disclosure relates to a method of increasing EImin in a patient diagnosed with sickle cell disease (SCD), comprising administering to the patient a sufficient amount of (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one or a pharmaceutically acceptable salt thereof.

In some embodiments, the disclosure relates to a method of improving RBC deformability in a patient diagnosed with sickle cell disease (SCD), comprising administering to the patient a sufficient amount of (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one or a pharmaceutically acceptable salt thereof.

In some embodiments, the disclosure relates to a method of reducing RBC turnover in a patient diagnosed with sickle cell disease (SCD), comprising administering to the patient a sufficient amount of (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one or a pharmaceutically acceptable salt thereof.

In some embodiments, the disclosure relates to a method of increasing RBC count in a patient diagnosed with sickle cell disease (SCD), comprising administering to the patient a sufficient amount of a PKR Activating Compound, e.g., (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one or a pharmaceutically acceptable salt thereof. In some embodiments, the disclosure relates to a method of increasing RBC count in a patient diagnosed with sickle cell disease (SCD), comprising administering to the patient a sufficient amount of (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one or a pharmaceutically acceptable salt thereof.

In some embodiments, the disclosure relates to a method of decreasing reticulocyte count in a patient diagnosed with sickle cell disease (SCD), comprising administering to the patient a sufficient amount of a PKR Activating Compound, e.g., (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one or a pharmaceutically acceptable salt thereof.

In some embodiments, the disclosure relates to a method of decreasing lactate dehydrogenase (LDH) concentration in a patient diagnosed with sickle cell disease (SCD), comprising administering to the patient a sufficient amount of (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one or a pharmaceutically acceptable salt thereof.

Compound 1 was evaluated in a multi-center, placebo-controlled Phase I trial in healthy volunteers and SCD patients ages 12 years and older. The healthy volunteer portion of the trial has been completed, and data has been presented at the 2019 American Society of Hematology meeting demonstrating the tolerability and proof of mechanism of Compound 1 in healthy volunteers. In RBCs of the healthy volunteers, Compound 1 demonstrated a reduction in 2,3-DPG and an increase in ATP, which provides confirmatory evidence of PKR activation in healthy RBCs. In addition, the reduction of 2,3-DPG correlated with increased oxygen affinity with single and multiple doses of Compound 1. A single dose cohort and a first multiple ascending dose, or MAD, cohort in SCD patients is ongoing. In the single dose cohort in SCD patients, a favorable tolerability profile and favorable biologic effects have been observed with evidence of pharmacodynamic activity translating into increased oxygen affinity and a shift in the Point of Sickling to lower oxygen tensions and improved membrane deformability of sickle RBCs. Furthermore, a second MAD cohort and a three-month open label extension in SCD patients are planned. Based on the results of this trial, global pivotal Phase II/III trial in SCD patients is planned. Clinical development of Compound 1 in pediatric SCD populations and other SCD patient populations in future trials is planned.

Methods of treating SCD also include administration of a therapeutically effective amount of a bioactive compound (e.g., a small molecule, nucleic acid, or antibody or other therapy) that reduces HgbS polymerization, for example by increasing HgbS affinity for oxygen. In some embodiments, a method of treating SCD comprises administering to a patient in need thereof a bioactive compound that reduces the percent of sickled cells in a murine model of sickle cell disease provided in Example 11 herein following 7 days of oral treatment with the compound. In some embodiments, the bioactive compound is any compound (including small molecules, nucleic acids, proteins or antibodies) that, in the murine model of sickle cell disease provided in Example 11, exhibits one or more of the following characteristics: (a) increases oxygen affinity to Hgb; (b) decreases p50; (c) decreases the percentage of RBCs that sickle at low oxygen pressures; (d) increases the time of a cell to sickle; and/or (e) increases Hgb by at least approximately 1 g/dL.

In some embodiments, Compound 1 is administered to a patient diagnosed with SCD, prior to, after or in combination with one or more additional SCD treatments administered to the patient. SCD treatments include curative therapies, disease modifying agents, symptomatic therapies administered as chronic prophylaxis or supportive care for acute crises.

The methods of treating SCD provided herein can offer greater protection against vaso-occlusive crises and hemolytic anemia, as compared to other therapies. Therefore, use of a PKR Activating Compound, such as Compound 1, provides a novel and improved therapeutic approach either alone or in combination with drugs that act through alternative mechanisms, such as hydroxyurea (HU). In some embodiments, Compound 1 is administered to a SCD patient who has previously received hydroxyurea (HU) or to a SCD patient undergoing HU treatment including patients who continue to receive HU when treated with Compound 1. HU, marketed under trade names including DROXIA by Bristol Myers Squibb Company, as well as in generic form, is approved for the treatment of anemia related to SCD, to reduce the frequency of VOCs and the need for blood transfusions. Hydroxyurea (HU) induces HgbF which interrupts the polymerization of HgbS, and thereby has activity in decreasing the onset of vaso-occlusive crises and pathological sequelae of SCD. While HU is in wide use as a backbone therapy for SCD, it remains only partially effective, and is associated with toxicity, such as myelosuppression and teratogenicity. Patients receiving HU still experience hemolysis, anemia, and vaso-occlusive crises, suggesting a need for more effective therapies, either as a replacement or in combination with HU. Beyond HU, therapeutic intervention is largely supportive care, aimed at managing the symptoms of SCD. For instance, blood transfusions help with the anemia and other SCD complications by increasing the number of normal RBCs and suppressing the synthesis of sickle RBCs. However, repeated transfusions lead to iron overload and the need for chelation therapies to avoid consequent tissue damage. In addition to these approaches, analgesic medications are used to manage pain. Many patients don't response to HU therapy, and even in responding patients, HU can lose efficacy over time. Although HU is considered to have an acceptable therapeutic index given the consequences of SCD, HU is underutilized due to safety concerns and side effects. HU and opioids are the standard non-curative treatments for chronic and acute care, respectively.

In some embodiments, a method of treating a patient diagnosed with SCD can include the steps of administering Compound 1 to the patient in combination with an antimetabolite such as HU, that is indicated to reduce the frequency of painful crises and to reduce the need for blood transfusions in patients with sickle cell anemia with recurrent moderate to severe painful crises. In some embodiments, the antimetabolite HU is administered with an initial dose of 15 mg/kg once daily, and the patient's blood count is monitored every two weeks. The dose of HU may be increased by 5 mg/kg/day every 12 weeks until a maximum tolerated dose or 35 mg/kg/day is reached if blood counts are in an acceptable range. The dose is not increased if blood counts are between the acceptable range and toxic. HU may be discontinued until hematologic recovery if blood counts are considered toxic. Treatment may then be resumed after reducing the dose by 2.5 mg/kg/day from the dose associated with hematological toxicity. The HU can be administered to the patient in hydroxyurea capsules, available for oral use as capsules containing 200 mg, 300 mg, and 400 mg hydroxyurea. Inactive ingredients with the HU can include citric acid, gelatin, lactose, magnesium stearate, sodium phosphate, titanium dioxide, and capsule colorants. Known pharmacologic effects of DROXIA that may contribute to its beneficial effects include increasing hemoglobin F levels in red blood cells (RBCs), decreasing neutrophils, increasing the water content of RBCs, increasing deformability of sickled cells, and altering the adhesion of RBCs to endothelium.

In some embodiments, Compound 1 is administered to a patient diagnosed with SCD who is also receiving L-glutamine for treatment of complications of SCD, and/or to a patient diagnosed with SCD who is has previously received L-glutamine for treatment of complications of SCD. Endari, marketed by Emmaus Life Sciences, Inc., is an oral powder form of L-glutamine approved to reduce severe complications associated with the disorder. L-glutamine is an amino acid indicated to reduce the acute complications of sickle cell disease in adult and pediatric patients 5 years of age and older. L-glutamine can be administered in an amount of 5 grams to 15 grams orally, twice daily based on body weight. Each dose of L-glutamine should be mixed in 8 oz. (240 mL) of cold or room temperature beverage or 4 oz. to 6 oz. of food before ingestion. L-glutamine is designated chemically as (S)-2-aminoglutaramic acid, L-glutamic acid 5-amide, or (S)-2, Oxidative stress phenomena are involved in the pathophysiology of SCD. Sickle red blood cells (RBCs) are more susceptible to oxidative damage than normal RBCs, which may contribute to the chronic hemolysis and vaso-occlusive events associated with SCD. The pyridine nucleotides, NAD+ and its reduced form NADH, play roles in regulating and preventing oxidative damage in RBCs. L-glutamine may improve the NAD redox potential in sickle RBCs through increasing the availability of reduced glutathione. 5-diamino-5-oxopentanoic acid. Following single-dose oral administration of L-glutamine at 0.1 g/kg, mean peak L-glutamine concentration was 1028 μM (or 150 mcg/mL) occurring approximately 30 minutes after administration. After an intravenous (IV) bolus dose, the volume of distribution was estimated to be approximately 200 mL/kg.

In some embodiments, Compound 1 is administered to a patient receiving supportive care for the management of VOCs. Supportive care for the management of painful VOCs entails the use of opioids or other pain medication.

In some embodiments, Compound 1 is administered to a patient diagnosed with SCD who has received (or is concurrently receiving) one or more therapies selected from the group consisting of voxelotor and crizanlizumab. In November 2019, the FDA approved voxelotor and crizanlizumab for the treatment of SCD.

In some embodiments, a method of treatment comprises administering Compound 1 to a patient diagnosed with SCD who has previously received a therapy for inhibiting polymerization of the HbS molecule. For example, Compound 1 can be administered to a SCD patient who has been treated with voxelotor. In some embodiments, Compound 1 is administered to a SCD patient in combination with voxelotor. FDA granted accelerated approval for voxelotor for the treatment of SCD in adults and children 12 years of age and older. Voxelotor is an oral therapy taken once daily and is the first approved treatment that directly inhibits HbS polymerization. Voxelotor is an oral small molecule therapy, which demonstrated improvement in total hemoglobin levels but failed to significantly decrease VOCs. Voxelotor is designed to reduce HbS polymerization by binding to the HbS molecule and stabilizing its binding to oxygen. Thus, the mechanism of voxelotor is specific for increasing HbS oxygenation to reduce HbS polymerization. While it achieved moderate increases in Hb content and reduction in hemolysis, this mechanism of action by itself is likely to be insufficient to effectively counter the significant anemia and blood vessel damage associated with this disease. Voxelotor is a hemoglobin S polymerization inhibitor indicated for the treatment of sickle cell disease in adults and pediatric patients 12 years of age and older. This indication is approved under accelerated approval based on increase in hemoglobin (Hb). Continued approval for this indication may be contingent upon verification and description of clinical benefit in confirmatory trial(s). The chemical name of voxelotor is: 2-hydroxy-6-((2-(1-isopropyl-1H-pyrazol-5-yl)pyridin-3-yl)methoxy)benzaldehyde. Voxelotor is a hemoglobin S polymerization inhibitor. Voxelotor is a hemoglobin S (HbS) polymerization inhibitor that binds to HbS with a 1:1 stoichiometry and exhibits preferential partitioning to red blood cells (RBCs). By increasing the affinity of Hb for oxygen, voxelotor demonstrates dose-dependent inhibition of HbS polymerization. Nonclinical studies suggest that voxelotor may inhibit RBC sickling, improve RBC deformability, and reduce whole blood viscosity. Voxelotor is absorbed into plasma and is then distributed predominantly into RBCs due to its preferential binding to Hb. The major route of elimination of voxelotor is by metabolism with subsequent excretion of metabolites into urine and feces. The PK are linear and voxelotor exposures increased proportionally with either single or multiple doses in whole blood, plasma, and RBCs. A high-fat, high-calorie meal increased voxelotor AUC by 42% and Cmax by 45% in whole blood relative to AUC and Cmax in the fasted state. Similarly, AUC increased by 42% and Cmax increased by 95% in plasma. In vitro and in vivo studies indicate that voxelotor is extensively metabolized through Phase I (oxidation and reduction), Phase II (glucuronidation) and combinations of Phase I and II metabolism. Oxidation of voxelotor is mediated primarily by CYP3A4, with minor contribution from CYP2C19, CYP2B6, and CYP2C9. The pharmacokinetic parameters of voxelotor were similar in pediatric patients 12 to <17 years and adults. Voxelotor steady state whole blood AUC and Cmax were 50% and 45% higher in HbSC genotype patients (n=11) compared to HbSS genotype (n=220) patients and voxelotor steady state plasma AUC and Cmax were 23% and 15% higher in HbSC genotype patients compared to HbSS genotype patients.

Another approach to treatment is exemplified by the monoclonal antibody crizanlizumab, a P-selectin blocking monoclonal antibody, which reduces VOCs but does not impact HbS polymerization. FDA approved crizanlizumab, to reduce the frequency of VOCs in adult and pediatric patients aged 16 years and older with SCD. Crizanlizumab is administered intravenously and binds to P-selectin, which is a cell adhesion protein that plays a central role in the multicellular interactions that can lead to vaso-occlusion. Crizanlizumab has shown benefit in reducing the number of VOCs but does not treat the underlying cause of SCD and is only administered through intravenous administration.

Blood transfusions are also used to treat SCD and can transiently bolster hemoglobin levels by adding functional RBCs. There are a number of limitations associated with this therapeutic approach, including limited patient access and serious complications such as iron overload.

Hematopoietic stem cell transplantation, or HSCT, is also an option for SCD patients, but this therapy is limited by toxic preconditioning regimens involving chemotherapy ablation, donor availability, and the need for post-transplant immunosuppression. Allogeneic HSCT is an invasive, potentially toxic, high-risk procedure limited by matched donor availability and significant procedure-associated morbidities. This treatment option is not commonly used given the difficulties of finding a suitable matched donor and the risks associated with the treatment, which include an approximately 5% mortality rate. HSCT is more commonly offered to pediatric patients with available sibling-matched donors. HSCT is typically recommended for only the most serious cases, and is largely offered only to children with sibling-matched donors. However, HSCT use can be severely limited by toxic preconditioning regimens, donor availability and the need for post-transplant immunosuppression.

Gene therapy is another SCD therapy also under investigation with promising preliminary results. Gene therapy and gene editing approaches in development provide promise for cures but are invasive, high-risk procedures that require toxic preconditioning regimens to ablate the bone marrow and make room for engineered cells that express either normal beta-globin or elevated levels of HbF. Furthermore, the long-term therapeutic durability of these approaches is unknown. These factors, in addition to the expected relatively high cost for treatment, may limit the use of gene therapy. A number of different therapeutic approaches are in development for patients with SCD. For example, a therapy called LentiGlobin is in clinical trial testing for the treatment of SCD. LentiGlobin is a one-time gene therapy treatment for SCD that aims to treat SCD by inserting a functional human beta-globin gene into the patient's own hematopoietic stem cells ex vivo and then transplanting the modified stem cell into the patient's bloodstream. Another therapy in development for treatment of SCD patients RVT-1801, a gene therapy, being evaluated in human clinical trials. Another therapy in development for treatment of SCD patients is BIVV-003, a gene editing cell therapy that modifies cells to produce functional RBCs using HbF.

The compound designated as IMR-687, a small molecule inhibitor of phosphodiesterase-9, is designed to increase production of HbF for the treatment of SCD. Another compound in development for treatment of SCD patients is EPI01, a small molecule designed to increase production of HbF, in clinical trials.

Treating Beta-Thalassemia with Compound 1

The administration of Compound 1 increased ATP in patients during the clinical trial of Example 12. Increasing ATP (and thereby improving membrane function) can benefit patients diagnosed with a thalassemia hemoglobinopathy. In some embodiments, Compound 1 can be administered for the treatment of beta thalassemia, which is a hemoglobinopathy that results from decreased or absent production of hemoglobin, thereby producing RBCs that have less oxygen carrying capacity than normal RBCs. Unlike SCD, beta thalassemia results from decreased or absent production of the beta subunit of hemoglobin, thereby producing RBCs that have less oxygen carrying capacity than normal RBCs. Further, the reduced levels of beta hemoglobin subunits result in an excess of alpha hemoglobin subunits, which form aggregates that can increase membrane damage and cause hemolysis. In some embodiments, Compound 1 can be administered to enhance the energy levels in beta thalassemia affected RBCs and enable the patients to tolerate the increased membrane damage and reduce hemolysis. The reduction in hemolysis can result in an increase in total hemoglobin that can improve symptoms.

Red blood cells (RBCs) in beta thalassemia patients have increased alpha-globin protein aggregates, free heme, and free iron that all cause an increase in the levels of toxic reactive oxygen species, which damage RBC membranes. Consequently, ATP is consumed more avidly in the RBCs of beta thalassemia patients, and this depletion of ATP stores is believed to be key to the reduced life span of RBCs and increased hemolysis in these patients. By increasing ATP levels in the RBCs of beta thalassemia patients, Compound 1 may reduce hemolysis and increase total body hemoglobin levels.

In some embodiments, Compound 1 can enhance the energy levels in beta thalassemia affected RBCs and enable the patients to tolerate the increased membrane damage and reduce hemolysis. The reduction in hemolysis can result in an increase in total hemoglobin that can improve symptoms.

Methods of treating beta thalassemia also include administration of a therapeutically effective amount of a bioactive compound (e.g., a small molecule, nucleic acid, or antibody or other therapy) that reduces HgbS polymerization, for example by increasing HgbS affinity for oxygen. In some embodiments, a method of treating beta thalassemia comprises administering to a patient in need thereof a bioactive compound that reduces the percent of sickled cells in a murine model of sickle cell disease provided in Example 11 herein following 7 days of oral treatment with the compound. In some embodiments, the bioactive compound is any compound (including small molecules, nucleic acids, proteins or antibodies) that, in the murine model of sickle cell disease provided in Example 11, exhibits one or more of the following characteristics: (a) increases oxygen affinity to Hgb in hypoxic conditions; (b) decreases p50 in hypoxic conditions; (c) decreases the percentage of RBCs that sickle at low oxygen pressures; (d) increases the time of a cell to sickle; and/or (e) increases Hgb by at least approximately 1 g/dL.

In some embodiments, methods of treatment comprise the step of administering Compound 1 to a patient diagnosed with previously confirmed hemoglobin genotype selected from the group consisting of Sβ0-thalassemia, or Sβ+-thalassemia, and wherein the patient is further characterized by one or more of the following: (1) age 12 to 65 years, (2) patients having had ≤6 vaso-occlusive crises (VOCs) within the past 12 months prior to receiving Compound 1, (3) no PRBC transfusion within 30 days of first receiving Compound 1; and (4) concomitant hydroxyurea use.

Patients with beta thalassemia are often classified into one of two groups; (i) transfusion dependent patients, and (ii) non-transfusion dependent patients. Transfusion dependent patients can require frequent blood transfusions, which may result in an overload of iron in tissues that can damage organs such as the liver, heart, and endocrine organs. As a consequence, iron depleting agents are used to minimize the consequences of iron overload. HSCT can be curative for beta thalassemia patients, but procedure related toxicity and donor availability limit this as a therapeutic option.

Until November 2019, there were no approved drug therapies for beta thalassemia in the United States. The standard of care for many patients with beta thalassemia has been frequent blood transfusions to manage anemia. A potentially curative therapy for beta thalassemia is HSCT, which is associated with serious risk and is limited to patients with a suitable donor.

In November 2019, luspatercept-aamt was approved by the FDA for the treatment of anemia in adult patients with beta thalassemia who are transfusion dependent (i.e., require regular RBC transfusions). Luspatercept-aamt, is a modified receptor protein that promotes RBC maturation and increases overall RBC production, but does not address other cell types implicated in beta thalassemia. Luspatercept-aamt is not indicated for use as a substitute for RBC transfusions in patients who require immediate correction of anemia. Luspatercept-aamt is dosed subcutaneously and is administered every three weeks in an outpatient setting. While studies suggest that luspatercept-aamt can reduce the number of transfusions that these patients may require and reduce iron loading, these patients remain transfusion dependent, and significant unmet needs remain for these patients.

Gene therapy approaches to increasing either beta-globin or HbF expression in autologous hematopoietic stem cells for transplantation are also in development but are limited by the need for marrow preconditioning and anticipated high cost. One gene therapy in development is the administration of autologous CD34⁺ cells encoding β^(A-T87Q)-globin gene, a gene therapy developed for the treatment of adult and adolescent patients with transfusion-dependent beta thalassemia and with certain genotypes.

Other therapeutic approaches in development for patients with transfusion-dependent beta thalassemia include Rivo-cel, a modified donor T cell therapy to be used in conjunction with HSCT; IMR-687, a small molecule inhibitor of phosphodiesterase-9; EPI01, a small molecule designed to increase production of HbF; OTL-300, an autologous ex vivo gene therapy for the treatment of transfusion-dependent beta thalassemia; ST-400, a genome-edited cell therapy approach designed to produce functional RBCs using HbF; CTX001, a gene editing approach to upregulate the expression of HbF, in patients with transfusion-dependent beta thalassemia; and gene control agents to activate gamma globin expression to induce the production of HbF for the treatment of beta thalassemia.

Methods of Preparing Compound 1 and Pharmaceutical Compositions

PKR Activating Compounds, such as 1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one, or a pharmaceutically acceptable salt thereof, are useful in pharmaceutical compositions for the treatment of patients. PKR Activating Compounds, such as Compound 1, or a pharmaceutically acceptable salt thereof, are useful in pharmaceutical compositions for the treatment of patients. The compositions comprising Compound 1, or a pharmaceutically acceptable salt thereof, can be obtained by certain processes also provided herein. The compositions comprising Compound 1, or a pharmaceutically acceptable salt thereof, can be obtained by certain processes also provided herein, such as the process provided in Example 1.

Pharmaceutical compositions can comprise Compound 1 and a pharmaceutically acceptable carrier. In some embodiments, a pharmaceutical composition comprising Compound 1 and Compound 2. In some embodiments, a provided pharmaceutical composition containing Compound 1 and Compound 2:

or a pharmaceutically acceptable salt thereof.

Representative “pharmaceutically acceptable salts” include, e.g., water-soluble and water-insoluble salts, such as the acetate, amsonate (4,4-diaminostilbene-2,2-disulfonate), benzenesulfonate, benzonate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium, calcium edetate, camsylate, carbonate, chloride, citrate, clavulariate, dihydrochloride, edetate, edisylate, estolate, esylate, fiunarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexafluorophosphate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, magnesium, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate, N-methylglucamine ammonium salt, 3-hydroxy-2-naphthoate, oleate, oxalate, palmitate, pamoate (1,1-methene-bis-2-hydroxy-3-naphthoate, einbonate), pantothenate, phosphate/diphosphate, picrate, polygalacturonate, propionate, p-toluenesulfonate, salicylate, stearate, subacetate, succinate, sulfate, sulfosalicylate, suramate, tannate, tartrate, teoclate, tosylate, triethiodide, and valerate salts.

In some embodiments, pharmaceutical compositions reported herein can be provided in a unit dosage form (e.g., capsule, tablet or the like).

Pharmaceutical compositions comprising a PKR Activating Composition containing a compound of Formula (I) can be formulated for oral administration (e.g., as a capsule or tablet). For example, Compound 1 can be combined with suitable compendial excipients to form an oral unit dosage form, such as a capsule or tablet, containing a target dose of Compound 1. The drug product can be prepared by first manufacturing Compound 1 as an active pharmaceutical ingredient (API), followed by spray drying with suitable polymer to obtain spray dried intermediate (SDD). SDD is then further processed by roller compaction/milling with intragranular excipients and blending with extra granular excipients. A Drug Product can contain the Compound 1 API and excipient components in Table 1 in a tablet in a desired dosage strength of Compound 1 (e.g., a 25 mg or 100 mg tablet formed from a Pharmaceutical Composition in Table 1). The blended material can be compressed to form tablets and then film coated.

In some embodiments, the API is an amorphous solid dispersion comprising Compound 1 and a polymer. In some embodiments, the polymer is selected from a group consisting of hydroxypropylmethyl cellulose (HPMC), hydroxypropylmethyl cellulose acetate succinate (HPMC AS), hydroxypropyl methyl cellulose phthalate (HPMCP), hydroxypropyl cellulose (HPC), ethylcellulose, cellulose acetate phthalate, polyvinylpyrrolidone (PVP), and a combination thereof. In some embodiments, the polymer is hydroxypropylmethyl cellulose (HPMC) or hydroxypropylmethyl cellulose acetate succinate (HPMC AS). In some embodiments, weight ratio of Compound 1 to the polymer in the amorphous solid dispersion is about 1:3. Table 1 provides an example of a tablet comprising a SDD obtained by the method of Example 1 and other components. In some examples, a tablet can weigh less than about 800 mg. In some examples, a tablet contains an amorphous Compound 1 API material in an amount providing about 10-40% by weight in the tablet of Compound 1 in addition to other ingredients such as a filler, dry binder, glidant and lubricant. In one example, a tablet contains 100 mg of Compound 1 in a tablet weight that is less than about 800 mg. In another example, a tablet contains 200 mg of Compound 1 in a tablet weight that is less than about 800 mg.

TABLE 1 Exemplary Pharmaceutical Compositions of Compound 1 for Oral Administration % Formulation (weight) Exemplary Component Intra- 50% 1:3 SDD of Granular Compound 1:HPMC AS-MG 30% Microcrystalline cellulose (Avicel PH 102)  5% Crospovidone (Kollidon CL-F) <5% Colloidal silicon dioxide (Aerosil 200) <1% Magnesium Stearate (Hyqual) Extra- 11% Microcrystalline cellulose (Avicel PH 200) Granular <5% Croscarmellose sodium (Ac-Di-Sol) <1% Magnesium Stearate (Hyqual)

In some embodiments, a provided composition containing a compound of Formula I comprises a mixture of (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one and (R)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one. In some embodiments, a provided composition containing a compound of Formula I is a mixture of Compound 1 and Compound 2 as part of a PKR Activating Composition. In some embodiments, a compound of Formula I is racemic. In some embodiments, a compound of Formula I consists of about 50% of Compound 1 and about 50% of Compound 2. In some embodiments, a compound of Formula I is not racemic. In some embodiments, a compound of Formula I does not consist of about 50% of Compound 1 and about 50% of Compound 2. In some embodiments, a compound of Formula I comprises about 99-95%, about 95-90%, about 90-80%, about 80-70%, or about 70-60% of Compound 1. In some embodiments, a compound of Formula I comprises about 99%, 98%, 95%, 90%, 80%, 70%, or 60% of Compound 1.

In some embodiments, a PKR Activating Composition comprises a mixture of Compound 1 and Compound 2. In some embodiments, a PKR Activating Composition comprises a mixture of Compound 1 and Compound 2, wherein the PKR Activating Composition comprises a therapeutically effective amount of Compound 1.

Compounds of Formula I, including Compound 1, can be obtained from a series of four reaction steps from commercially available starting materials, as outlined in Example 1. Commercially available 7-bromo-2H,3H-[1,4]dioxino[2,3-b]pyridine was treated with a mixture of n-butyl lithium and dibutylmagnesium followed by sulfuryl chloride to give sulfonyl chloride 3. Treatment of 3 with tert-butyl 1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrole-2-carboxylate in the presence of triethylamine (TEA) afforded Boc-protected monosulfonamide 4. Compound 4 was then de-protected in the presence of trifluoroacetic acid (TFA) to give 5, the free base of the monosulfonamide. The last step to generate Compound 1 (Example 1, Step 5) or Compound 1 and Compound 2 (Example 1, Step 4) was an amide coupling of 5 and tropic acid in the presence of 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU).

In some embodiments, pharmaceutical compositions reported herein can be provided in an oral dosage form. In some embodiments, the pharmaceutical composition is orally administered in any orally acceptable dosage form. In some embodiments, an oral dosage form of a PKR Activating Compound be a capsule. In some embodiments, an oral dosage form of a PKR Activating Compound is a tablet. In some embodiments, an oral dosage form comprises one or more fillers, disintigrants, lubricants, glidants, anti-adherents and/or anti-statics. In some embodiments, an oral dosage form is prepared via dry blending. In some embodiments, an oral dosage form is a tablet and is prepared via dry granulation.

Additional Embodiments

Methods of treatment (e.g., by activating PKR) can comprise administering to a subject in need thereof a therapeutically effective amount of (i) a compound disclosed herein, or a pharmaceutically acceptable salt thereof or (ii) a pharmaceutical composition comprising a compound disclosed herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. The PKR Activating Compound can be administered orally, for the treatment of diseases or conditions that therapeutically benefit from the administration of a compound that activates PKR, including hemoglobinopathies such as SCD or beta-thalassemia. In some embodiments, Compound 1 can be administered orally, for the treatment of diseases or conditions that therapeutically benefit from the administration of a compound that activates PKR, such as SCD or beta-thalassemia. Compound 1 is a potent activator of PKR and may improve RBC metabolism, function and survival. Compound 1 may also be useful for improving both hemoglobin levels and decreasing the rate of VOCs.

In some embodiments, a method of treating a disease associated with modulation of PKR comprises administering a therapeutically effective amount of a compound disclosed herein. In some embodiments, a method of treating pyruvate kinase deficiency (PKD) comprises administering a therapeutically effective amount of a compound disclosed herein. In some embodiments, a method of treating PKD-associated hemolytic anemia comprises administering a therapeutically effective amount of a compound disclosed herein.

Methods of treatment can comprise administering to a subject in need thereof a therapeutically effective amount of (i) a PKR Activating Compound (e.g., a compound disclosed herein), or a pharmaceutically acceptable salt thereof, or (ii) a PKR Activating Composition (e.g., a pharmaceutical composition comprising a compound disclosed herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier). The pharmaceutical composition may be orally administered in any orally acceptable dosage form.

One aspect of the disclosure relates to methods of treating a patient comprising the administration of a therapeutically effective amount of Compound 1 or a pharmaceutically acceptable salt thereof, such as a patient diagnosed with a hemoglobinopathy. In some embodiments, the patient is diagnosed with a hemoglobinopathy, such as Sickle Cell Disease or beta-thalassemia.

In some embodiments, Compound 1 can be administered orally, once-daily, for the treatment of a hemoglobinopathy, such as or beta-thalassemia or SCD. In some embodiments, Compound 1 can be administered orally, once-daily, for the treatment of SCD. In some embodiments, Compound 1 can be administered orally, once-daily, for the treatment of beta-thalassemia. Compound 1 is a potent activator of PKR and may improve RBC metabolism, function and survival. Compound 1 may also be useful for improving both hemoglobin levels and decreasing the rate of VOCs. Methods of treating a patient diagnosed with SCD can include administering to the patient in need thereof a therapeutic compound targeting reduction of deoxy-HgbS, which may or may not directly improve RBC membrane integrity. Compound 1 has been shown to decrease 2,3-DPG and increase ATP, and reduced cell sickling has been demonstrated in disease models. Accordingly, in some embodiments, the methods of treatment can address not only sickling, but also hemolysis and anemia.

In some embodiments, Compound 1 can be administered orally, once-daily, for the treatment of beta-thalassemia. Compound 1 is a potent activator of PKR and may improve RBC metabolism, function and survival. Compound 1 may also be useful for improving both hemoglobin levels. Methods of treating a patient diagnosed with beta-thalassemia can include administering to the patient in need thereof a therapeutic compound targeting reduction of deoxy-HgbS, which may or may not directly improve RBC membrane integrity. Compound 1 has been shown to decrease 2,3-DPG and increase ATP, and reduced cell sickling has been demonstrated in disease models. Accordingly, in some embodiments, the methods of treatment can address not only sickling, but also hemolysis and anemia.

Methods of treating a patient diagnosed with sickle cell disease, and PKR Activating Compounds for use in such methods, can include administering to the patient the PKR Activating Compound (e.g., a composition comprising one or more compounds of Formula I, such as Compound 1 or a mixture of Compound 1 and Compound 2) in an amount sufficient to reduce 2,3-DPG levels in the patient's red blood cells. Methods of treating a patient diagnosed with beta thalassemia, and PKR Activating Compounds for use in such methods, can include administering to the patient the PKR Activating Compound (e.g., a composition comprising one or more compounds of Formula I, such as Compound 1 or a mixture of Compound 1 and Compound 2) in an amount sufficient to reduce 2,3-DPG levels in the patient's red blood cells. In some embodiments, the amount is sufficient to reduce 2,3-DPG levels by at least 30% after 24 hours, or greater (e.g., reducing 2,3-DPG levels in the patient's red blood cells by at least 40% after 24 hours). In some embodiments, the amount is sufficient to reduce 2,3-DPG levels by 30-50% after 24 hours. In some embodiments, the amount is sufficient to reduce 2,3-DPG levels by 40-50% after 24 hours. In some embodiments, the amount is sufficient to reduce 2,3-DPG levels by at least 25% after 12 hours. In some embodiments, the amount is sufficient to reduce 2,3-DPG levels by 25-45% after 12 hours. In some embodiments, the amount is sufficient to reduce 2,3-DPG levels by at least 15% after 6 hours. In some embodiments, the amount is sufficient to reduce 2,3-DPG levels by 15-30% after 6 hours. In some embodiments, the amount is sufficient to reduce 2,3-DPG levels by at least 40% on day 14 of treatment. In some embodiments, the amount is sufficient to reduce 2,3-DPG levels by 40-60% on day 14 of treatment. In some embodiments, the amount is sufficient to reduce 2,3-DPG levels by at least 50% on day 14 of treatment. In some embodiments, the amount is sufficient to reduce 2,3-DPG levels by 50-60% on day 14 of treatment.

Methods of treating a patient diagnosed with sickle cell disease, and PKR Activating Compounds for use in such methods, can also include administering to the patient the PKR Activating Compound (e.g., a composition comprising one or more compounds of Formula I, such as Compound 1 or a mixture of Compound 1 and Compound 2) in a daily amount sufficient to increase the patient's ATP blood levels. Methods of treating a patient diagnosed with beta thalassemia, and PKR Activating Compounds for use in such methods, can also include administering to the patient the PKR Activating Compound (e.g., a composition comprising one or more compounds of Formula I, such as Compound 1 or a mixture of Compound 1 and Compound 2) in a daily amount sufficient to increase the patient's ATP blood levels. In some embodiments, the amount is sufficient to increase ATP blood levels by at least 40% on day 14 of treatment, or greater (e.g., at least 50% on day 14 of treatment). In some embodiments, the amount is sufficient to increase ATP blood levels by 40-65% on day 14 of treatment. In some embodiments, the amount is sufficient to increase ATP blood levels by at least 50% on day 14 of treatment, or greater (e.g., at least 50% on day 14 of treatment). In some embodiments, the amount is sufficient to increase ATP blood levels by 50-65% on day 14 of treatment.

A therapeutically effective amount of a Compound 1 can be administered to a patient in need thereof in a pharmaceutical composition. For example, administration of a therapeutically effective amount of a PKR Activating Compound can include administration of a total of about 25 mg-1,500 mg of Compound 1 each day, in single or divided doses. In some embodiments, Compound 1 is administered to patients diagnosed with SCD in total once daily (QD) doses of 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, and/or higher if tolerated (e.g., 250 mg, 300 mg, 500 mg, 600 mg, 1000 mg, and/or 1500 mg). In some embodiments, a human dose of 80 to 130 mg of Compound 1 is administered once daily (QD) to a patient in need thereof (e.g., a patient diagnosed with SCD). In some embodiments, a PKR Activating Compound is administered in an amount of 400 mg per day (e.g., 400 mg QD or 200 mg BID). In some embodiments, Compound 1 or a pharmaceutically acceptable salt thereof is administered in an amount of 400 mg per day (e.g., 400 mg QD or 200 mg BID). In some embodiments, Compound 1 or a pharmaceutically acceptable salt thereof is administered in an amount of 400 mg per day (e.g., 400 mg QD or 200 mg BID). In some embodiments, a PKR Activating Compound is administered in an amount of 700 mg per day (e.g., 700 mg QD or 350 mg BID). In some embodiments, Compound 1 or a pharmaceutically acceptable salt thereof is administered in an amount of 700 mg per day (e.g., 700 mg QD or 350 mg BID). In some embodiments, Compound 1 or a pharmaceutically acceptable salt thereof is administered in an amount of 700 mg per day (e.g., 700 mg QD or 350 mg BID). In some embodiments, a PKR Activating Compound is administered in an amount of 100 mg, 200 mg, 400 mg, 600 mg, 700 mg, 1100 mg, or 1500 mg per day, in single or divided doses. In some embodiments, Compound 1 or a pharmaceutically acceptable salt thereof is administered in an amount of 100 mg, 200 mg, 400 mg, 600 mg, 700 mg, 1100 mg, or 1500 mg per day, in single or divided doses. In some embodiments, Compound 1 or a pharmaceutically acceptable salt thereof is administered in an amount of 100 mg, 200 mg, 400 mg, 600 mg, 700 mg, 1100 mg, or 1500 mg per day, in single or divided doses.). In some embodiments, Compound 1 or a pharmaceutically acceptable salt thereof is administered in an amount of 200 mg per day (QD).

In some embodiments, a daily dose of between 100 mg to 1500 mg of a PKR Activating Compound is administered to humans. In some embodiments, a daily dose of between 100 mg to 1500 mg of Compound 1 is administered to humans. In some embodiments, a daily dose of between 100 mg to 1500 mg of Compound 1 is administered to humans. In particular, a total daily dose of 100 mg-600 mg of a PKR Activating Compound can be administered to humans (including, e.g., a dose of 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, or 600 mg, per day, in single or divided doses). In particular, a total daily dose of 100 mg-600 mg of Compound 1 can be administered to humans (including, e.g., a dose of 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, or 600 mg, per day, in single or divided doses). In particular, a total daily dose of 100 mg-600 mg of Compound 1 can be administered to humans (including, e.g., a dose of 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, or 600 mg, per day, in single or divided doses). In some embodiments, a daily dose of 400 mg (e.g., 400 mg QD or 200 mg BID) of a PKR Activating Compound is administered to humans. In some embodiments, a daily dose of 400 mg (e.g., 400 mg QD or 200 mg BID) of Compound 1, or a pharmaceutically acceptable salt thereof, is administered to humans. In some embodiments, a daily dose of 400 mg (e.g., 400 mg QD or 200 mg BID) Compound 1 is administered to humans.

In some embodiments, a total daily dose of 100 mg-600 mg of (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one is administered to the patient per day. In some embodiments, the method can comprise administering (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one to the patient in a total dose and dose interval selected from the group consisting of 100 mg BID, 200 mg BID, 300 mg BID and 400 mg QD. In some embodiments, a total of 300 mg QD of (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one is administered to a patient diagnosed with SCD. In some embodiments, a total of 300 mg QD of (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one is administered to a patient diagnosed with beta-thalassemia. A method of treating a patient diagnosed with Sickle Cell Disease (SCD) can comprise repeatedly administering to the patient in need thereof a total of 300 mg QD of (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one.

In some examples, a pharmaceutical composition comprising Compound 1 can be used in a method of treating a patient diagnosed with sickle cell disease, the method comprising administering to the patient 400 mg of Compound 1 or a pharmaceutically acceptable salt thereof, once per day (QD)

In some examples, a pharmaceutical composition comprising Compound 1 can be used in a method of treating a patient diagnosed with sickle cell disease, the method comprising administering to the patient 300 mg of Compound 1 or a pharmaceutically acceptable salt thereof once per day (QD)

In some examples, a pharmaceutical composition comprising Compound 1 can be used in a method of treating a patient diagnosed with sickle cell disease, the method comprising administering to the patient 200 mg of Compound 1 or a pharmaceutically acceptable salt thereof, once per day (QD)

In some embodiments, the present disclosure provides PKR Activating Compounds of Formula I:

or a pharmaceutically acceptable salt thereof. In some embodiments, a PKR Activating Compound is 1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one.

The compound of Formula I is preferably Compound 1:

or a pharmaceutically acceptable salt thereof. In some embodiments, a compound of Formula I is (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one. In some examples, Compound 1 is a stable, crystalline substance. In some examples, Compound 1 is an amorphous substance.

The pharmaceutical composition comprising Compound 1 can be administered to the patient throughout a medically appropriate course of treatment, which can be a series of consecutive days for multiple consecutive weeks. In some embodiments, (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one is administered to the patient over multiple consecutive days.

Some embodiments provide an oral, once-daily dosage form (e.g., a tablet or capsule) comprising Compound 1 for use in a therapy for increasing hemoglobin oxygen affinity by reducing 2,3-DPG blood concentrations, increasing hemoglobin levels and/or increasing intracellular ATP, without significant effects affecting sex hormones (e.g., without aromatase inhibition activity) or inducing its own metabolism upon repeat daily administration throughout a course of treatment.

Some embodiments provide an oral, once-daily dosage form (e.g., a tablet or capsule) comprising Compound 1 for use in a therapy for increasing hemoglobin oxygen affinity without significant effects affecting sex hormones (e.g., without aromatase inhibition activity) or inducing its own metabolism upon repeat daily administration throughout a course of treatment.

Some embodiments provide an oral, once-daily dosage form (e.g., a tablet or capsule) comprising Compound 1 for use in a therapy for reducing 2,3-DPG blood concentrations, without significant effects affecting sex hormones (e.g., without aromatase inhibition activity) or inducing its own metabolism upon repeat daily administration throughout a course of treatment.

Some embodiments provide an oral, once-daily dosage form (e.g., a tablet or capsule) comprising Compound 1 for use in a therapy for increasing hemoglobin levels, without significant effects affecting sex hormones (e.g., without aromatase inhibition activity) or inducing its own metabolism upon repeat daily administration throughout a course of treatment.

Some embodiments provide an oral, once-daily dosage form (e.g., a tablet or capsule) comprising Compound 1 for use in a therapy for increasing intracellular ATP, without significant effects affecting sex hormones (e.g., without aromatase inhibition activity) or inducing its own metabolism upon repeat daily administration throughout a course of treatment.

Some embodiments provide an oral, once-daily dosage form (e.g., a tablet or capsule) comprising Compound 1 for use in a therapy without significant effects affecting sex hormones (e.g., without aromatase inhibition activity) or inducing its own metabolism upon repeat daily administration throughout a course of treatment.

In other embodiments, the disclosure relates to each of the following numbered embodiments:

1. A composition comprising a PKR Activating Compound of Formula I, or a pharmaceutically acceptable salt thereof:

2. The composition of embodiment 1, wherein the compound of Formula I is Compound 1, or a pharmaceutically acceptable salt thereof:

3. The composition of embodiment 2, wherein the composition comprises a mixture of Compound 1 and Compound 2, or a pharmaceutically acceptable salt thereof:

4. The composition of embodiment 1, comprising the compound: 1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one. 5. The composition of any one of embodiments 1-4, formulated as an oral unit dosage form. 6. A method of treating a patient diagnosed with a sickle cell disease (SCD), the method comprising administering to the patient in need thereof a therapeutically effective amount of a pharmaceutical composition comprising (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one, or a pharmaceutically acceptable salt thereof. 7. The method of embodiment 6, wherein the method comprises oral administration of the pharmaceutical composition comprising (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one, as the only PKR Activating Compound in the pharmaceutical composition. 8. A method of treating a patient diagnosed with a sickle cell disease (SCD), the method comprising administering to the patient in need thereof a therapeutically effective amount of a pharmaceutical composition comprising Compound 1:

or a pharmaceutically acceptable salt thereof. 9. A composition comprising a compound of Formula I obtainable by a process comprising the step of converting compound 5 into a compound of Formula I in a reaction described as Step 4:

10. The composition of embodiment 9, wherein the process further comprises first obtaining the compound 5 from a compound 4 by a process comprising Step 3:

11. The composition of embodiment 10, wherein the process further comprises first obtaining the compound 4 from a compound 3 by a process comprising Step 2:

12. The composition of embodiment 11, wherein the process further comprises first obtaining the compound 3 from a process comprising Step 1:

13. A method of treating a patient diagnosed with sickle cell disease (SCD), the method comprising administering to the patient in need thereof a therapeutically effective amount of a PKR Activating Compound having an AC₅₀ value of less than 1 μM using the Luminescence Assay described in Example 2. 14. The method of embodiment 13, wherein the PKR Activating Compound is Compound 1. 15. The method of any one of embodiments 13-14, wherein the PKR Activating Compound is orally administered to the patient in need thereof.

16. The use of Compound 1:

or a pharmaceutically acceptable salt thereof, for the treatment of patients diagnosed with sickle cell disease (SCD). 17. The use of a PKR Activating Compound having an AC₅₀ value of less than 1 μM using the Luminescence Assay described in Example 2, in the treatment of patients diagnosed with sickle cell disease. 18. The method of any one of embodiments 6-8 or 13-15, comprising the administration of Compound 1 once per day. 19. The method of any one of embodiments 6-8 or 13-15, comprising the administration of a total of 25 mg-1,500 mg of Compound 1 each day. 20. The method of any one of embodiments 18-19, comprising the administration of a total of 25 mg-130 mg of Compound 1 each day. 21. A method of treating a patient diagnosed with SCD, comprising the administration to the patient of a therapeutically effective amount of a PKR Activating Compound, wherein the PKR Activating Compound, in the murine model of sickle cell disease provided in Example 11, exhibits one or more of the following characteristics: (a) increases oxygen affinity to Hgb in hypoxic conditions; (b) decreases p50 in hypoxic conditions; (c) decreases the percentage of RBC that sickle at low oxygen pressures; (d) increases the time of a cell to sickle; and/or (e) increases Hgb by at least approximately 1 g/dL. 22. The method of embodiment 21, wherein the PKR Activating Compound is an antibody. 23. The method of embodiment 21, wherein the PKR Activating Compound is a protein. 24. The method of embodiment 21, wherein the PKR Activating Compound is a nucleic acid. 25. The method of embodiment 21, wherein the PKR Activating Compound is a DNA nucleic acid. 26. The method of embodiment 21, wherein the PKR Activating Compound is a RNA nucleic acid.

In other embodiments, the disclosure relates to each of the following numbered embodiments:

1. A PKR Activating Compound for use in a method of treating a patient diagnosed with sickle cell disease (SCD), comprising administering to the patient the PKR Activating Compound in a therapeutically effective amount, wherein the PKR Activating Compound is a compound of Formula I:

or a pharmaceutically acceptable salt thereof, having an AC₅₀ value of less than 1 μM using the Luminescence Assay described in Example 2. 2. The PKR Activating Compound of embodiment 1, wherein the PKR Activating Compound is Compound 1:

or a pharmaceutically acceptable salt thereof. 3. The PKR Activating Compound of embodiment 1, wherein the PKR Activating Compound is Compound 1:

4. The PKR Activating Compound of embodiment 3, wherein the PKR Activating Compound is administered in an amount of 25-1500 mg per day. 5. The PKR Activating Compound of embodiment 3, wherein the PKR Activating Compound is administered once daily in an amount of 250 mg, 300 mg, 500 mg, 600 mg, 1000 mg, or 1500 mg per day. 6. The PKR Activating Compound of embodiment 3, wherein the PKR Activating Compound is administered once daily in an amount of 100 mg per day. 7. The PKR Activating Compound of embodiment 3, wherein the PKR Activating Compound is administered once daily in an amount of 600 mg per day. 8. The PKR Activating Compound of embodiment 3, wherein the PKR Activating Compound is administered once per day. 9. The PKR Activating Compound of embodiment 3, wherein the PKR Activating Compound is orally administered to the patient. 10. The PKR Activating Compound of embodiment 3, wherein Compound 1 is the only PKR Activating Compound administered to the patient. 11. A PKR Activating Compound for use in a method of treating a patient diagnosed with sickle cell disease, comprising administering to the patient the PKR Activating Compound in an amount sufficient to reduce 2,3-DPG levels in the patient's red blood cells by at least 30% after 24 hours, wherein the PKR Activating Compound is a compound of Formula I.

or a pharmaceutically acceptable salt thereof, having an AC₅₀ value of less than 1 μM using the Luminescence Assay described in Example 2. 12. The PKR Activating Compound of embodiment 11, wherein the PKR Activating Compound is Compound 1:

or a pharmaceutically acceptable salt thereof. 13. The PKR Activating Compound of embodiment 1, wherein the PKR Activating Compound is Compound 1:

14. The PKR Activating Compound of embodiment 13, wherein Compound 1 is the only PKR Activating Compound administered to the patient. 15. The PKR Activating Compound of any one of embodiments 11-14, wherein the PKR Activating Compound is orally administered to the patient. 16. The PKR Activating Compound of any one of embodiments 11-15, wherein the PKR Activating Compound is administered once per day. 17. The PKR Activating Compound of any one of embodiments 11-16, wherein the PKR Activating Compound is administered in an amount sufficient to reduce 2,3-DPG levels in the patient's red blood cells by at least 40% after 24 hours. 18. The PKR Activating Compound of any one of embodiments 11-17, wherein the PKR Activating Compound is administered in a daily amount sufficient to increase the patient's ATP blood levels by at least 40% on day 14 of treatment. 19. The PKR Activating Compound of any one of embodiments 11-15, wherein the PKR Activating Compound is administered in an amount of 100 mg, 200 mg, 400 mg, 600 mg, 700 mg, 1100 mg, or 1500 mg per day. 20. The PKR Activating Compound of any one of embodiments 11-15, wherein the PKR Activating Compound is administered in an amount of 200 mg per day. 21. The PKR Activating Compound of embodiment 20, wherein the PKR Activating Compound is administered in an amount of 200 mg per day once per day (QD). 22. The PKR Activating Compound of embodiment 20, wherein the PKR Activating Compound is administered in an amount of 100 mg per day twice per day (BID). 23. The PKR Activating Compound of any one of embodiments 11-15, wherein the PKR Activating Compound is administered in an amount of 400 mg per day. 24. The PKR Activating Compound of embodiment 23, wherein the PKR Activating Compound is administered in an amount of 400 mg once per day (QD). 25. The PKR Activating Compound of embodiment 23, wherein the PKR Activating Compound is administered in an amount of 200 mg twice per day (BID). 26. The PKR Activating Compound of any one of embodiments 11-15, wherein the PKR Activating Compound is administered in an amount of 600 mg per day. 27. The PKR Activating Compound of embodiment 26, wherein the PKR Activating Compound is administered in an amount of 300 mg twice per day (BID). 28. The PKR Activating Compound of any one of embodiments 11-15, wherein the PKR Activating Compound is administered in an amount of 700 mg per day. 29. The PKR Activating Compound of embodiment 28, wherein the PKR Activating Compound is administered in an amount of 700 mg once per day (QD). 30. The PKR Activating Compound of embodiment 28, wherein the PKR Activating Compound is administered in an amount of 350 mg twice per day (BID).

In other embodiments, the disclosure relates to each of the following numbered embodiments:

31. A pharmaceutical composition comprising Compound 1 and a pharmaceutically acceptable carrier:

for use in a method of treating a patient diagnosed with a sickle cell disease (SCD), the method comprising administering to the patient in need thereof a total of 25 mg-1,500 mg of Compound 1 per day. 32. The composition of embodiment 31, wherein the method comprises the administration of Compound 1 in a single dose once per day. 33. The composition of embodiment 31, wherein the method comprises the administration of Compound 1 in a divided dose each day. 34. The composition of any one of embodiments 31-33, wherein the composition is orally administered to the patient. 35. The composition of any one of embodiments 31-34, wherein the composition is formulated as an oral unit dosage form. 36. A method of treating a patient diagnosed with a sickle cell disease (SCD), the method comprising orally administering to the patient in need thereof a total of 25 mg-1,500 mg per day of (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one in a pharmaceutical composition. 37. A method of treating a patient diagnosed with a sickle cell disease (SCD), the method comprising orally administering to the patient in need thereof a total of 25 mg-1,500 mg of Compound 1 per day:

in a pharmaceutical composition comprising Compound 1 and a pharmaceutically acceptable carrier. 38. The method of any one of embodiments 36-37, wherein (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one is the only PKR Activating Compound in the pharmaceutical composition. 39. The method of any one of embodiments 36-38, comprising the administration of Compound 1 in a single dose once per day. 40. The method of any one of embodiments 36-38, comprising the administration of Compound 1 in a divided dose each day. 41. A pharmaceutical composition comprising a PKR Activating Compound for use in a method of treating a patient diagnosed with sickle cell disease, comprising administering to the patient the PKR Activating Compound in an amount sufficient to reduce 2,3-DPG levels in the patient's red blood cells by at least 30% after 24 hours, wherein the PKR Activating Compound is a compound of Formula I:

or a pharmaceutically acceptable salt thereof, having an AC50 value of less than 1 μM using the Luminescence Assay described in Example 2. 42. The composition of embodiment 41, wherein the PKR Activating Compound is Compound 1:

or a pharmaceutically acceptable salt thereof. 43. The composition of embodiment 42, wherein Compound 1 is the only PKR Activating Compound administered to the patient. 44. The composition of any one of embodiments 41-43, wherein the PKR Activating Compound is orally administered to the patient. 45. The composition of any one of embodiments 41-44, wherein the PKR Activating Compound is administered once per day. 46. The composition of any one of embodiments 41-45, wherein the PKR Activating Compound is administered in an amount sufficient to reduce 2,3-DPG levels in the patient's red blood cells by at least 40% after 24 hours. 47. The composition of any one of embodiments 41-46, wherein the PKR Activating Compound is administered in a daily amount sufficient to increase the patient's ATP blood levels by at least 40% on day 14 of treatment. 48. The composition of any one of embodiments 41-45, wherein the PKR Activating Compound is administered in an amount of 100 mg, 200 mg, 400 mg, 600 mg, 700 mg, 1100 mg, or 1500 mg per day. 49. The composition of any one of embodiments 41-44, wherein the PKR Activating Compound is administered in an amount of 200 mg per day. 50. The composition of any one of embodiments 41-44, wherein the PKR Activating Compound is orally administered in an amount of 200 mg per day once per day (QD). 51. The composition of any one of embodiments 41-44, wherein the PKR Activating Compound is orally administered in an amount of 100 mg per day twice per day (BID). 52. The composition of any one of embodiments 41-44, wherein the PKR Activating Compound is administered in an amount of 400 mg per day in a single or divided dose. 53. The composition of embodiment 41, wherein the PKR Activating Compound is orally administered in an amount of 400 mg once per day (QD). 54. The composition of any one of embodiments 41-44, wherein the PKR Activating Compound is orally administered in an amount of 200 mg twice per day (BID). 55. The composition of any one of embodiments 41-44, wherein the PKR Activating Compound is administered in an amount of 700 mg per day in a single or divided dose. 56. The composition of any one of embodiments 41-44, wherein the PKR Activating Compound is administered in an amount of 700 mg once per day (QD). 57. The composition of any one of embodiments 41-44, wherein the PKR Activating Compound is orally administered in an amount of 350 mg twice per day (BID).

In other embodiments, the disclosure relates to each of the following embodiments:

A method for increasing oxygen affinity of sickle hemoglobin (HbS) in vivo in a patient in need thereof which method comprises administering to said patient a sufficient amount of (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one or a pharmaceutically acceptable salt thereof. In some embodiments, the administration of a single dose of (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one or a salt thereof increases the oxygen affinity of said HbS in the patient.

A method for inhibiting sickling of HbS in a patient diagnosed with Sickle Cell Disease, (SCD), which method comprises administering to said patient a sufficient amount of a composition comprising (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one or a pharmaceutically acceptable salt thereof.

A method of treating a patient diagnosed with Sickle Cell Disease (SCD), comprising administering to said patient a therapeutically effective single dose of (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one or a pharmaceutically acceptable salt thereof, such that the patient experiences a left shift in the point of sickling (PoS) with an increase in the EImin after 24 hours.

A method of treating a patient diagnosed with Sickle Cell Disease (SCD), comprising administering to a patient (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one or a pharmaceutically acceptable salt thereof, in an amount effective to increase oxygen affinity of HbS.

A method of treatment, comprising administering to a patient (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one or a pharmaceutically acceptable salt thereof, in an amount effective to increase oxygen affinity of HbA.

A method of treatment, comprising administering to a patient (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one or a pharmaceutically acceptable salt thereof, in an amount effective to increase oxygen affinity of HbS. In some embodiments, the patient is diagnosed with Sickle Cell Disease or beta-thalassemia.

A method of treatment, comprising administering to a patient (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one or a pharmaceutically acceptable salt thereof, in an amount effective to result in a left shift in the point of sickling (PoS) with an increase in the EImin in the patient. In some embodiments, the patient is diagnosed with Sickle Cell Disease or beta-thalassemia.

A method of increasing Hb concentration in a patient diagnosed with sickle cell disease (SCD), comprising administering to the patient a sufficient amount of (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one or a pharmaceutically acceptable salt thereof.

A method of reducing RBC turnover in a patient diagnosed with sickle cell disease (SCD), comprising administering to the patient a sufficient amount of (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one or a pharmaceutically acceptable salt thereof.

A method of decreasing lactate dehydrogenase (LDH) concentration in a patient diagnosed with sickle cell disease (SCD), comprising administering to the patient a sufficient amount of (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one or a pharmaceutically acceptable salt thereof.

A method of increasing RBC count in a patient diagnosed with sickle cell disease (SCD), comprising administering to the patient a sufficient amount of (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one or a pharmaceutically acceptable salt thereof.

A method of decreasing reticulocyte count in a patient diagnosed with sickle cell disease (SCD), comprising administering to the patient a sufficient amount of (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one or a pharmaceutically acceptable salt thereof.

A method of reducing point of sickling (POS) in a patient diagnosed with sickle cell disease (SCD), comprising administering to the patient a sufficient amount of (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one or a pharmaceutically acceptable salt thereof.

A method of increasing EImin in a patient diagnosed with sickle cell disease (SCD), comprising administering to the patient a sufficient amount of (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one or a pharmaceutically acceptable salt thereof.

A method of improving RBC deformability in a patient diagnosed with sickle cell disease (SCD), comprising administering to the patient a sufficient amount of (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one or a pharmaceutically acceptable salt thereof.

A method of improving RBC membrane function in a patient diagnosed with sickle cell disease (SCD), comprising administering to the patient a sufficient amount of (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one or a pharmaceutically acceptable salt thereof. In some embodiments, said improving RBC membrane function comprises improving RBC membrane response to an osmotic gradient, as evidenced by a shift toward normal in Omin and Ohyper.

In some or any of the above embodiments, a total daily dose of 100 mg-600 mg of (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one is administered to the patient per day.

In some or any of the above embodiments, the (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one is administered to the patient over multiple consecutive days.

In some or any of the above embodiments, administering (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one to the patient in a total dose and dose interval selected from the group consisting of 100 mg BID, 200 mg BID, 300 mg BID and 400 mg QD.

In some or any of the above embodiments, a total of 300 mg QD of (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one is administered to the patient, wherein the patient is diagnosed with SCD.

In some or any of the above embodiments, a total of 300 mg QD of (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one is administered to the patient, wherein the patient is diagnosed with beta-thalassemia.

A method of treating a patient diagnosed with Sickle Cell Disease (SCD) comprising repeatedly administering to the patient in need thereof a total of 300 mg QD of (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one.

A method of treating a patient diagnosed with a hemoglobinopathy, the method comprising administering a PKR Activating Compound in an amount effective to increase oxygen affinity of HbS in the patient or to provide a left shift in the point of sickling (PoS) with an increase in the EImin in the patient, or a combination thereof.

In some or any of the above embodiments, the hemoglobinopathy is Sickle Cell Disease or beta-thalassemia.

A method of treating a patient diagnosed with Sickle Cell Disease (SCD) comprising repeatedly administering to the patient in need thereof a dose of 400 mg QD of (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one.

A method of treating a patient diagnosed with Sickle Cell Disease (SCD) comprising repeatedly administering to the patient in need thereof a dose of 300 mg QD of (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one.

In some or any of the above embodiments, the (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one is administered to the patient each day for at least 7 days.

In some or any of the above embodiments, the (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one is administered to the patient each day for at least 14 days.

In some or any of the above embodiments, the (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one is administered to the patient each day for at least 28 days.

In some or any of the above embodiments, the (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one is administered to the patient each day for at least 60 days.

In some or any of the above embodiments, the (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one is administered to the patient each day for at least 120 days.

In some or any of the above embodiments, the patient had from 1 to 10 vasoocclusive crisis (VOC) events within 12 months prior to enrollment and baseline hemoglobin (Hb) ≥5.5 to ≤10.5 g/dL prior to treatment with (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one.

In some or any of the above embodiments, the patient has not received red blood cell (RBC) transfusions within 60 days or erythropoietin within 28 days, does not have renal insufficiency, does not have uncontrolled liver disease, is not pregnant, and is not lactating, at the time of treatment with (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one or the PKR Activating Compound.

In some or any of the above embodiments, the patient is treated with the (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one until the patient has a Hb response rate defined as a Hb increase of >1 g/dL from baseline compared to a patient treated with placebo.

In some or any of the above embodiments, the patient is treated with the (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one once daily for at least 24 consecutive weeks.

In some or any of the above embodiments, the patient is treated with the (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one twice daily for at least 24 consecutive weeks.

A method comprising administering to a patient diagnosed with a hemoglobinopathy a therapeutically effective amount of (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one, the therapeutically effective amount being effective to provide one or more effects in the patient in need thereof, selected from the group consisting of: increase oxygen affinity of sickle hemoglobin (HbS) in the patient; and inhibit the sickling of HbS in the patient.

A method of increasing oxygen affinity of sickle hemoglobin (HbS) or inhibiting the sickling of HbS in a patient diagnosed with Sickle Cell Disease, the method comprising administering to the patient in need thereof a therapeutically effective amount of (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one.

In some or any of the above embodiments, the (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one is orally administered.

In some or any of the above embodiments, the (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one is administered once daily.

In some or any of the above embodiments, the (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one is administered for at least 24 consecutive weeks.

In some or any of the above embodiments, a total of 300 mg per day of (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one is administered to the patient each day.

A method of treatment comprising the step of administering to a patient diagnosed with a hemoglobinopathy a therapeutically effective amount of (R)-2-Hydroxy-2-phenyl-1-(5-(pyridin-2-ylsulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)ethan-1-one, or a pharmaceutically acceptable salt thereof.

In some or any of the above embodiments, the hemoglobinopathy is Sickle Cell Disease, PKD or beta-thalassemia.

In some or any of the above embodiments, the patient has a hemoglobin genotype selected from the group consisting of Hgb SS, Hgb Sβ+-thalassemia, Hgb Sβ0-thalassemia, and Hgb SC.

In some or any of the above embodiments, the hemoglobin genotype is Hgb SS.

In some or any of the above embodiments, the hemoglobin genotype was confirmed by hemoglobin electrophoresis or genotyping.

In some or any of the above embodiments, the patient has not started hydroxyurea (HU) therapy within 90 days prior to said administering.

The method of any one of claims 1-55, wherein the patient has not received crizanlizumab within 14 days prior to said administering.

In some or any of the above embodiments, the patient has not received voxelotor within 7 days prior to said administering.

In some or any of the above embodiments, the patient has not received a red blood cell transfusion within 30 days prior to said administering.

In some or any of the above embodiments, the patient has a hemoglobin level of about 7.0 g/dL to about 10.5 g/dL.

In some or any of the above embodiments, the patient is ≥12 years of age.

In some or any of the above embodiments, the patient is <18 years of age.

In some or any of the above embodiments, the patient is <12 years of age.

In some or any of the above embodiments, the patient is <6 years of age.

In some or any of the above embodiments, the patient is <3 years of age.

In some or any of the above embodiments, the method comprises improving anemia or complications associated with anemia in a patient with Hgb SS or Hgb SB0-thalassemia.

In some or any of the above embodiments, the patient is being treated with a concurrent medication that is a CYP substrate.

In some or any of the above embodiments, the concurrent medication is a sensitive CYP substrate.

A pharmaceutical composition comprising the compound (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one or a pharmaceutically acceptable salt thereof for use in increasing the oxygen affinity of HgbA in a patient, by administering to the patient the pharmaceutical composition in an amount effective to increase the oxygen affinity of the HgbA as measured by a decrease in the p50 measured 24 hours after the administration of the pharmaceutical composition to the patient.

A pharmaceutical composition comprising the compound (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one or a pharmaceutically acceptable salt thereof for use in increasing the oxygen affinity of HgbS in a patient diagnosed with Sickle Cell Disease (SCD), by administering to the patient the pharmaceutical composition in an amount effective to increase the oxygen affinity of the HgbS as measured by a decrease in the p50 measured 24 hours after the administration of the pharmaceutical composition to the patient.

A pharmaceutical composition comprising the compound (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one or a pharmaceutically acceptable salt thereof for use in increasing the oxygen affinity of HgbS in a patient diagnosed with Sickle Cell Disease (SCD), by administering to the patient the pharmaceutical composition in an amount effective to reduce 2,3-diphosphoglycerate (2,3-DPG) in the blood of the patient measured 24 hours after the administration of the pharmaceutical composition to the patient.

A pharmaceutical composition comprising the compound (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one or a pharmaceutically acceptable salt thereof for use in treating a patient diagnosed with a hemolytic anemia, wherein the patient's hemolytic anemia was previously confirmed by hemoglobin electrophoresis or genotyping indicating one of the following hemoglobin genotypes: Hgb SS, Hgb Sβ+-thalassemia, Hgb Sβ0-thalassemia, or Hgb SC.

In some embodiments, the disclosure relates to:

-   -   1. The compound         (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one         for use in a single daily (QD) administration of 200 mg to 1,000         mg of         (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one         a human subject.     -   2. The compound of embodiment 1, for use in reducing the 2,3-DPG         concentration in the blood of the human subject for 24-72 hours         after administering the compound once daily to the subject for         14 consecutive days.     -   3. The compound of embodiment 1, for use in increasing the ATP         concentration in the blood of the human subject for 24-72 hours         after administering the compound once daily to the subject for         14 consecutive days.     -   4. The compound of embodiment 1, for use in decreasing the LDH         concentration in the blood of the human subject for 24-72 hours         after administering the compound once daily to the subject for         14 consecutive days.     -   5. The compound of embodiment 1, for use in increasing the         oxygen affinity (p50) of RBCs in the blood of the human subject         for 24 hours after administering the compound once to the         subject.     -   6. The compound of embodiment 1, for use in activating PKR         without inhibiting aromatase.     -   7. The compound of embodiment 1, for use in activating PKR         without CYP inhibition or induction.     -   8. The compound of embodiment 1, for use in simultaneously         activating PKR, increasing ATP, decreasing 2,3-DPG and         increasing oxygen affinity (p50) in the blood of the subject for         72 hours after administering the compound to the subject.     -   9. The compound of any one of embodiments 1-8, wherein the         subject is diagnosed with Sickle Cell Disease (SCD).     -   10. The compound of embodiment 9, for use in the treatment of a         pediatric patient diagnosed with Sickle Cell Disease (SCD).     -   11. The compound of embodiment 10, wherein the pediatric SCD         patient is younger than age 12.     -   12. The compound of embodiment 10, wherein the pediatric SCD         patient is between the ages of 12 and 18.     -   13. The compound of embodiment 10, wherein the pediatric SCD         patient is younger than age 2.     -   14. The compound         (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one         for use in the treatment of Sickle Cell Disease in a subject         having a Hgb SS or Hgb SC hemoglobin genotypes.     -   15. The compound         (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one         for use in increasing the oxygen affinity of red blood cells of         a subject having a normal hemoglobin genotype selected from the         group consisting of HbA, HbA1, HbA2, HbE, HbF, HbS, HbC, HbH,         and HbM, and having HbF <2% of total hemoglobin.

The present disclosure enables one of skill in the relevant art to make and use the inventions provided herein in accordance with multiple and varied embodiments. Various alterations, modifications, and improvements of the present disclosure that readily occur to those skilled in the art, including certain alterations, modifications, substitutions, and improvements are also part of this disclosure. Accordingly, the foregoing description and drawings are by way of example to illustrate the discoveries provided herein.

EXAMPLES

As the enzyme that catalyzes the last step of glycolysis, PKR underlies reactions that directly impact the metabolic health and primary functions of RBCs. The following Examples demonstrate how PKR activation by Compound 1 impacts RBCs. The primary effect of Compound 1 on RBCs is a decrease in 2,3-DPG that is proposed to reduce Hgb sickling and its consequences on RBCs and oxygen delivery to tissues. Compound 1 also increases ATP, which may provide metabolic resources to support cell membrane integrity and protect against loss of deformability and increased levels of hemolysis in SCD. With the combination of effects Compound 1 has on RBCs, it is likely to reduce the clinical sequelae of sickle Hgb and provide therapeutic benefits for patients with SCD.

The PKR Activating Compound designated Compound 1 was prepared as described in Example 1, and tested for PKR activating activity in the biochemical assay of Example 2.

The biological enzymatic activity of PKR (i.e., formation of ATP and/or pyruvate) was evaluated in enzyme and cell assays with Compound 1, as described in Example 3 and Example 4, respectively. Results from enzyme assays show that Compound 1 is an activator of recombinant wt-PKR and mutant PKR, (e.g., R510Q), which is one of the most prevalent PKR mutations in North America. PKR exists in both a dimeric and tetrameric state, but functions most efficiently as a tetramer. Compound 1 is an allosteric activator of PKR and is shown to stabilize the tetrameric form of PKR, thereby lowering the K_(m) (the Michaelis-Menten constant) for PEP.

Similarly, results from assays with RBCs from human patients with SCD showed that treatment with Compound 1 caused a shift in p50 (PO₂ at 50% hemoglobin saturation) and that this shift was related to increased oxygen affinity in the presence of Compound 1 (Example 5). Furthermore, Compound 1 decreased sickling under severe hypoxic conditions. Taken together the data suggest that Compound 1 can reduce the clinical consequences of sickled cells by decreasing cell sickling through an increase in oxygen affinity that comes from PKR activation.

In vivo testing in mice (Examples 8 and 11) demonstrated PKR activation in wt mice, and provided an evaluation of effects on RBCs and Hgb in a murine model of SCD. Compound 1 was well tolerated up to the highest dose tested, and exposures increased in a dose-proportional manner. Levels of 2,3-DPG were reduced by >30% for doses ≥120 mg/kg Compound 1 (AUC from 0 to 24 hours (AUC₀₋₂₄>5200 hr ng/mL) and levels of ATP were increased by >40% for ≥60 mg/kg Compound 1 (AUC₀₋₂₄>4000 hr ng/mL). In two studies with a murine model of SCD, increased oxygen affinity, decreased sickling, and increased Hgb were observed ex vivo in RBCs from mice following 7 days of oral (chow) administration of Compound 1. Taken together, the data support exposure-related therapeutic benefits of Compound 1 for treatment of SCD.

Compound 1 activates wild type as well as G332S and R510Q variants of pyruvate kinase R with an AC50 of less than 1 micromolar in the Luminescence Assay of Example 2. Compound 1 activates wild type and R510Q pyruvate kinase with an AC50 value of less than 0.1 micromolar in the Enzyme Assay of Example 3. Compound 1 activates wt-PKR in mature human erythrocytes in a concentration dependent manner with an EC50 of less than 0.5 micromolar in the Cell Assay of Example 4.

Compound 1 increases the oxygen affinity of Hgb in red blood cells (RBCs) from both healthy subjects (HgbA) and in patients diagnosed with Sickle Cell Disease (HgbS), as measured by a reduction in p50, the oxygen level at which 50% of the hemoglobin is oxygenated. Reduction in p50 represents an increase in oxygen affinity. A shift in p50 representing increased oxygen affinity is observed in RBCs after 1 hour and maintained for at least 3 hours from blood obtained from patients diagnosed with SCD (Example 5). Mixing Compound 1 with RBCs from both healthy volunteers and patients diagnosed with SCD results in increased oxygen affinity measured by a reduction in the p50 values measured for both types of RBCs (Example 6).

Compound 1 reduces cell sickling under severe hypoxic conditions of 2% oxygen, providing up to about 16% percent protection defined as the level of activity in treated cells, normalized to the level of activity in untreated cells after exposure to the severe hypoxic conditions as measured in Example 5. Compound 1 reduces the point of sickling (PoS) in RBCs from patients diagnosed with SCD, when measured by improved RBC deformability and a decrease in elongation index (EI) in the presence of Compound 1 as described in Example 7. In addition, Compound 1 increased oxygen affinity to Hgb under hypoxic conditions, decreased p50, decreased the percentage of RBCs that sickled at low oxygen pressures, and increased the time to sickle in a murine SCD mouse model expressing human HgbS (Example 11).

Example 1: Synthesis of Compounds of Formula I

The PKR Activating Compound 1 was obtained by the method described herein. Compound 1 has a molecular weight of 457.50 Da.

Step 1. 2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl chloride (3)

Into a 100 mL round-bottom flask purged and maintained with an inert atmosphere of nitrogen was placed a solution of n-BuLi in hexane (2.5 M, 2 mL, 5.0 mmol, 0.54 equiv) and a solution of n-Bu₂Mg in heptanes (1.0 M, 4.8 mL, 4.8 mmol, 0.53 equiv). The resulting solution was stirred for 10 min at RT (20° C.). This was followed by the dropwise addition of a solution of 7-bromo-2H,3H-[1,4]dioxino[2,3-b]pyridine (2 g, 9.26 mmol, 1.00 equiv) in tetrahydrofuran (16 mL) with stirring at −10° C. in 10 min. The resulting mixture was stirred for 1 h at −10° C. The reaction mixture was slowly added to a solution of sulfuryl chloride (16 mL) at −10° C. The resulting mixture was stirred for 0.5 h at −10° C. The reaction was then quenched by the careful addition of 30 mL of saturated ammonium chloride solution at 0° C. The resulting mixture was extracted with 3×50 mL of dichloromethane. The organic layers were combined, dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The residue was purified by silica gel column chromatography, eluting with ethyl acetate/petroleum ether (1:3). This provided 1.3 g (60%) of 2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl chloride as a white solid. LCMS m/z: calculated for C₇H₆ClNO₄S: 235.64; found: 236 [M+H]⁺.

Step 2. tert-Butyl 5-[2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl]-H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrole-2-carboxylate (4)

Into a 100-mL round-bottom flask was placed 2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl chloride (1.3 g, 5.52 mmol, 1.00 equiv), tert-butyl 1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrole-2-carboxylate (1.16 g, 5.52 mmol), dichloromethane (40 mL), and triethylamine (1.39 g, 13.74 mmol, 2.49 equiv). The solution was stirred for 2 h at 20° C., then diluted with 40 mL of water. The resulting mixture was extracted with 3×30 mL of dichloromethane. The organic layers were combined, dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The residue was purified by silica gel column chromatography, eluting with dichloromethane/methanol (10:1). This provided 1.2 g (53%) of tert-butyl 5-[2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl]-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrole-2-carboxylate as a yellow solid. LCMS m/z: calculated for C₁₈H₂₃N₃O₆S: 409.46; found: 410 [M+H]⁺.

Step 3. 2-[2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl]-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrole (5)

Into a 100-mL round-bottom flask was placed tert-butyl 5-[2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl]-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrole-2-carboxylate (1.2 g, 2.93 mmol, 1.00 equiv), dichloromethane (30 mL), and trifluoroacetic acid (6 mL). The solution was stirred for 1 h at 20° C. The resulting mixture was concentrated under vacuum. The residue was dissolved in 10 mL of methanol and the pH was adjusted to 8 with sodium bicarbonate (2 mol/L). The resulting solution was extracted with 3×10 mL of dichloromethane. The organic layers were combined, dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The crude product was purified by silica gel column chromatography, eluting with dichloromethane/methanol (10:1). This provided 650 mg (72%) of 2-[2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl]-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrole as a yellow solid. LCMS m/z: calculated for C₁₃H₁₅N₃O₄S: 309.34; found: 310 [M+H]⁺.

Step 4. (S)-1-(5-[2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl]-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl)-3-hydroxy-2-phenylpropan-1-one (1) and (R)-1-(5-[2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl]-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl)-3-hydroxy-2-phenylpropan-1-one (2)

Into a 100 mL round-bottom flask was placed 2-[2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl]-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrole (150 mg, 0.48 mmol, 1.00 equiv), 3-hydroxy-2-phenylpropanoic acid (97 mg, 0.58 mmol, 1.20 equiv), dichloromethane (10 mL), HATU (369 mg, 0.97 mmol, 2.00 equiv) and DIEA (188 mg, 1.46 mmol, 3.00 equiv). The resulting solution was stirred overnight at 20° C. The reaction mixture was diluted with 20 mL of water and was then extracted with 3×20 mL of dichloromethane. The organic layers were combined, dried over anhydrous sodium sulfate, filtered and concentrated under vacuum. The residue was purified by prep-TLC eluted with dichloromethane/methanol (20:1) and further purified by prep-HPLC (Column: XBridge C18 OBD Prep Column, 100 Å, 5 μm, 19 mm×250 mm; Mobile Phase A: water (10 mmol/L NH₄HCO₃), Mobile Phase B: MeCN; Gradient: 15% B to 45% B over 8 min; Flow rate: 20 mL/min; UV Detector: 254 nm). The two enantiomers were separated by prep-Chiral HPLC (Column, Daicel CHIRALPAK® IF, 2.0 cm×25 cm, 5 m; mobile phase A: DCM, phase B: MeOH (hold 60% MeOH over 15 min); Flow rate: 16 mL/min; Detector, UV 254 & 220 nm). This resulted in peak 1 (2, Rt: 8.47 min) 9.0 mg (4%) of (R)-1-(5-[2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl]-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl)-3-hydroxy-2-phenylpropan-1-one as a yellow solid; and peak 2 (1, Rt: 11.83 min) 10.6 mg (5%) of (S)-1-(5-[2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl]-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl)-3-hydroxy-2-phenylpropan-1-one as a yellow solid.

(1): ¹H NMR (400 MHz, DMSO-d₆) δ 8.13 (d, J=2.0 Hz, 1H), 7.61 (d, J=2.0 Hz, 1H), 7.31-7.20 (m, 5H), 4.75 (t, J=5.2 Hz, 1H), 4.50-4.47 (m, 2H), 4.40-4.36 (m, 1H), 4.32-4.29 (m, 2H), 4.11-3.87 (m, 8H), 3.80-3.77 (m, 1H), 3.44-3.41 (m, 1H). LC-MS (ESI) m/z: calculated for C₂₂H₂₃N₃O₆S: 457.13; found: 458.0 [M+H]⁺.

(2): ¹H NMR (400 MHz, DMSO-d₆) δ 8.13 (d, J=2.0 Hz, 1H), 7.60 (d, J=2.0 Hz, 1H), 7.31-7.18 (m, 5H), 4.75 (t, J=5.2 Hz, 1H), 4.52-4.45 (m, 2H), 4.40-4.36 (m, 1H), 4.34-4.26 (m, 2H), 4.11-3.87 (m, 8H), 3.80-3.78 (m, 1H), 3.44-3.43 (m, 1H). LC-MS (ESI) m/z: calculated for C₂₂H₂₃N₃O₆S: 457.13; found: 458.0 [M+H]⁺.

Step 5. (S)-1-(5-[2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl]-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl)-3-hydroxy-2-phenylpropan-1-one (1)

Alternatively, Compound 1 can be synthesized using the procedure described here as Step 5. A solution of 7-((3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)sulfonyl)-2,3-dihydro-[1,4]dioxino[2,3-b]pyridine (130.9 mg, 0.423 mmol) in DMF (2.5 ml) was cooled on an ice bath, then treated with (S)-3-hydroxy-2-phenylpropanoic acid (84.8 mg, 0.510 mmol), HATU (195.5 mg, 0.514 mmol), and DIEA (0.30 mL, 1.718 mmol) and stirred at ambient temperature overnight. The solution was diluted with EtOAc (20 mL), washed sequentially with water (20 mL) and brine (2×20 mL), dried (MgSO₄), filtered, treated with silica gel, and evaporated under reduced pressure. The material was chromatographed by Biotage MPLC (10 g silica gel column, 0 to 5% MeOH in DCM) to provide a white, slightly sticky solid. The sample was readsorbed onto silica gel and chromatographed (10 g silica gel column, 0 to 100% EtOAc in hexanes) to provide (2S)-1-(5-[2H,3H-[1,4]dioxino[2,3-b]pyridine-7-sulfonyl]-1H,2H,3H,4H,5H,6H-pyrrolo[3,4-c]pyrrol-2-yl)-3-hydroxy-2-phenylpropan-1-one (106.5 mg, 0.233 mmol, 55% yield) as a white solid.

Step 6. Preparing a Spray Dried Dispersion of Compound 1

A Spray Dried Dispersion (SDD) of Compound 1 was prepared. The SDD was made up of Compound 1 and a polymer (Hydroxypropylmethyl Cellulose AS-MG) at a 1:3 ratio. Compound 1 and the polymer were dissolved in organic solvents (Dichloromethane and Methanol) and spray dried to obtain amorphous an amorphous drug substance.

A spray solution was prepared at 7.8% solids content (1:3 Compound 1:HPMC AS-MG) in 80:20 DCM:Methanol per Table A. An API correction factor of 0.966 was used to prepare the spray solution. The spray solution was prepped by adding DCM and Methanol to a 36L stainless steel mixing vessel. HPMC AS-MG was added to the solvent system while mixing with a top down mixer at a medium vortex. Compound 1 was then added to the solution. The solution had a yellow/brown clear appearance.

TABLE A Component Formulation % Weight, g Compound 1  2.00% 595.0 HPMC AS-MG  5.81% 1724.3 DCM 73.75% 21896.0 Methanol 18.44% 5474.0 Total 100.0% 29689.3 Correction Factor: 0.9660

A Mobile Minor spray-drying apparatus was setup per Table B and warmed up for approximately one hour prior to spraying. Wash solution (80:20 DCM:Methanol) was sprayed prior to the active solution to allow the nozzle to equilibrate. The Compound 1 active solution was sprayed per the settings in Table B. The spray-dried dispersion was dried overnight (˜20 hours) in a Shel Vacuum Oven at 50° C. and −25 in Hg vacuum under a nitrogen purge at 15 scfh. The resulting spray-dried dispersion was confirmed to be dry by GC analysis. This run generated approximately 2.1 kg of spray-dried dispersion.

TABLE B Parameter Set Point Inline Filter Swagelok 140 μm Stainless Steel Nozzle 0.3 mm, 60° Angle Inlet Air Flow 80 kg/hr Inlet Air Temperature 104° C. Pump Stroke Length 5.70 mm Nozzle Pressure 600 psi Feed Rate (g/min) 184 g/min Outlet Temp (° C.) 36 Set Condenser Air Temp (° C.) −10 Actual Condenser Air −3 Temp (° C.) Chiller Temp (° C.) −20 Feed Temp Ambient

Example 2: Biochemical Assay for Identification of PKR Activating Activity

PKR Activating Compounds can be identified with the biochemical Luminescence Assay of Example 2. The PKR activating activity of a series of chemical compounds was evaluated using the Luminescence Assay below, including compounds designated Compound 1, and Compound 2, or mixtures thereof.

For each tested compound, the ability to activate PKR was determined using the following Luminescence Assay. The effect of phosphorylation of adenosine-5′-diphosphate (ADP) by PKR is determined by the Kinase Glo Plus Assay (Promega) in the presence or absence of FBP (D-fructose-1,6-diphosphate; BOC Sciences, CAS: 81028-91-3) as follows. Unless otherwise indicated, all reagents are purchased from Sigma-Aldrich. All reagents are prepared in buffer containing 50 mM Tris-HCl, 100 mM KCl, 5 mM MgCl₂, and 0.01% Triton X100, 0.03% BSA, and 1 mM DTT. Enzyme and PEP (phosphoenolpyruvate) are added at 2× to all wells of an assay-ready plate containing serial dilutions of test compounds or DMSO vehicle. Final enzyme concentrations for PKR(wt), PKR(R510Q), and PKR(G332S) are 0.8 nM, 0.8 nM, and 10 nM respectively. Final PEP concentration is 100 μM. The Enzyme/PEP mixture is incubated with compounds for 30 minutes at RT before the assay is initiated with the addition of 2×ADP and KinaseGloPlus. Final concentration of ADP is 100 μM. Final concentration of KinaseGloPlus is 12.5%. For assays containing FBP, that reagent is added at 30 μM upon reaction initiation. Reactions are allowed to progress for 45 minutes at RT until luminescence is recorded by the BMG PHERAstar FS Multilabel Reader. The compound is tested in triplicate at concentrations ranging from 42.5 μM to 2.2 nM in 0.83% DMSO. AC₅₀ measurements were obtained by the standard four parameter fit algorithm of ActivityBase XE Runner (max, min, slope and AC₅₀). The AC₅₀ value for a compound is the concentration (μM) at which the activity along the four parameter logistic curve fit is halfway between minimum and maximum activity.

As set forth in Tables 2 and 3 below, AC₅₀ values are defined as follows: ≤0.1 M (+++); >0.1 μM and ≤1.0 μM (++); >1.0 μM and ≤40 μM (+); >40 μM (0).

TABLE 2 Luminescence Assay Data AC₅₀ AC₅₀ AC₅₀ Compound (PKRG332S) (PKRR510Q) (WT) 1 ++ +++ +++ 2 + + +

TABLE 3 Additional Luminescence Assay Data AC₅₀ AC₅₀ Compound Structure (PKRG332S) (PKRR510Q) 6

++ + 7

0 0 8

0 0

Compounds and compositions described herein are activators of wild type PKR and certain PKR mutants having lower activities compared to the wild type. Such mutations in PKR can affect enzyme activity (catalytic efficiency), regulatory properties, and/or thermostability of the enzyme. One example of a PKR mutation is G332S. Another example of a PKR mutation is R510Q.

Example 3: Enzyme Assays of a PKR Activating Compound

The ability of Compound 1 to activate PKR in enzyme-based assays was measured. Significant increases in PKR activity as measured by Vmax, a biochemical measure of the maximal rate of enzyme activity, of up to 1.8-fold were observed under certain physiologic conditions as shown in FIG. 7 . In particular, activation of PKR by different concentrations of Compound 1 was evaluated for phosphoenolpyruvate, or PEP, concentrations at or below the K_(m).

The effect of 2 μM Compound 1 on maximum velocity (V_(max)) and PEP K_(m) (Michaelis-Menten constant, i.e., the concentration of PEP at which v=½v_(max)) was evaluated for wt-PKR and PKR-R510Q. Tests were conducted in the presence and absence of fructose-1,6-bisphosphate (FBP), a known allosteric activator of PKR. Assessments were made up to 60 min at RT, and Vmax and PEP K_(m) were calculated. The effect of Compound 1 on Vmax ranged from no effect to a modest increase (see FIG. 7 for a representative curve). Compound 1 consistently reduced the PEP K_(m), typically by ˜2 fold, for wt-PKR and PKR-R510Q in the presence or absence of FBP (Table 4), demonstrating that Compound 1 can enhance the rate of PKR at physiological concentrations of PEP.

TABLE 4 Effect of Compound 1 on PKR Enzyme Kinetic Parameters No FBP 30 μM FBP Kinetic 2 μM 2 μM Enzyme Parameter^(a) DMSO Compound 1 DMSO Compound 1 WT- V_(max) 1.00 1.14 1.19 1.16 PKR PEP K_(m) 4.84 2.44 1.98 1.00 PKR V_(max) 1.54 1.56 1.00 1.29 R510Q PEP K_(m) 6.20 1.70 2.01 1.00 ^(a)All values in Table 4 are normalized to 1.00, relative to the other values in the same row.

Activation of wt-PKR and PKR-R510Q by different concentrations of Compound 1 was evaluated for PEP concentrations at or below K_(m). Compound 1 increased the rate of ATP formation, with AC₅₀ values ranging from <0.05 to <0.10 μM and a range of <2.0 to <3.0 maximum-fold activation (ie, <200% to <300%) (Table 5). Representative data from PKR-R510Q showed that the effect was concentration dependent (FIG. 8 ).

TABLE 5 Activation of PKR Wild and Mutant Types by Compound 1 PK Enzyme Maximum-fold Activation AC₅₀ (μM) WT-PKR <2.0 <0.05 PKR R510Q <3.0 <0.10

Example 4: Cell Assays of a PKR Activating Compound

The activation of wt-PKR by Compound 1 in mature human erythrocytes ex vivo was evaluated in purified RBCs purchased from Research Blood Components. Cells treated with Compound 1 for 3 hr in glucose-containing media were washed, lysed, and assayed using a Biovision Pyruvate Kinase Assay (K709-100). The assay was repeated multiple times to account for donor-to-donor variability and the relatively narrow dynamic range. Mean maximum activation increase (Max-Min) was <100% and mean 50% effective concentration (EC₅₀) was <125 nM (Table 6). wt-PKR was activated in a concentration-dependent manner (FIG. 9 ).

TABLE 6 Wild Type PKR Activation in Human Red Blood Cells Treated with Compound 1 Replicate Max-Min (%) EC₅₀ (nM) 1 <125 <250 2 <150 <150 3 <100 <50 4 <50 <50 Mean <100 <125

Mouse RBCs were isolated fresh from whole blood using a Ficoll gradient and assayed with methods similar to those used in the human RBCs assays. Maximum activation increase, and EC₅₀ values were comparable to the effects in human RBCs (Table 7).

TABLE 7 Effect of Compound 1 on PKR Activation in Mouse Red Blood Cells Replicate Max-Min (%) EC₅₀ (nM) 1 <50 <125 2 <100 <125 Mean <100 <125

Example 5: Ex Vivo Pharmacology of a PKR Activating Compound

Red blood cells from SCD patients were used to evaluate the effects of Compound 1 on Hgb affinity for oxygen (i.e., oxygen affinity) and sickling under hypoxic conditions. Cells were incubated at 37° C. for 1, 2, and 3 hr with HEPES buffered saline (HBS) (untreated), HBS+dimethyl sulfoxide (DMSO) (vehicle), or 10 μM Compound 1. To assess oxygen dissociation, Hgb oxygen equilibrium curves were collected during deoxygenation.

Hemoglobin saturation was shifted to the left in cells treated with Compound 1 and not in untreated or 0.5% DMSO-treated cells (FIGS. 10 and 11 ). The increased oxygen affinity corresponded to a significant (but limited) shift in p50 from 29 to 25 mmHg after 1 hr that was maintained until at least 3 hr, the last time point evaluated (Table 8). Notably, oxygen affinity in the first 2 hr of incubation was not affected by DMSO.

TABLE 8 Effect of Compound 1 on Hemoglobin Saturation^(a) Hemoglobin Saturation Incubation DMSO Compound Time (hr) Untreated^(a) (0.5%) 1 (10 μM) 1 1.18 1.18 1.05 2 1.18 1.00 3 1.30 1.02 ^(a)All values in Table 8 are normalized to 1.00, relative to the other values. ^(b)Untreated cells are washed RBCs at 40% hematocrit in media without incubation.

At each PO₂, the average shift in Hgb saturation in the cells treated with Compound 1 was most pronounced around 25 mmHg, compared to a normal PO₂ of 26.7 mmHg (FIG. 12 ). Therefore, the shift in oxygen affinity occurred at oxygen tensions that are relevant for sickling. At 2 hr, Hgb saturation is approximately 10% higher compared to DMSO-treated cells. There is a clear difference between the cells treated with Compound 1 and those treated with DMSO at lower PO₂ (approximately 10 mmHg at 1% to 2% oxygen) even at 1 hr.

Compound 1 (10 μM) reduced cell sickling under severe hypoxic conditions of 2% oxygen (PO₂ of <20 mmHg) for up to 20 min (Table 9). The percent protection (i.e., the level of activity in treated cells, normalized to the level of activity in untreated cells after exposure to severe hypoxic conditions) reached a maximum of 16% at 15 min under hypoxic conditions (FIG. 13 ) and remained at 15% at the last time point measured.

TABLE 9 Effect of Compound 1 on Sickling of Human SCD Cells in Hypoxic Conditions Time in Hypoxic Net % of Sickled Cells Conditions DMSO Compound 1 (min) (0.5%) (10 μM) % Protection 0 7 10 2 41 47 −15 5 57 49 14 10 61 54 11 15 68 57 16 20 71 60 15

Example 6: Increases in Hemoglobin Oxygen Affinity (p50) in Mixing Compound 1 in In Vitro Studies with RBCs from Healthy and SCD Donors

As illustrated in FIG. 14 , mixing Compound 1 with RBCs from healthy donors and SCD donors increases RBC oxygen affinity in HbA and HbS RBCs, respectively, as reflected by the leftward shift in the curves, which can be characterized by the oxygen level at which 50% of hemoglobin is oxygenated, or p50. In vitro incubation with Compound 1 increases oxygen affinity in HbA RBCs, consistent with clinical results in studies with healthy volunteers, and increases oxygen affinity in HbS RBCs, indicating that the PKR enzyme in sickle RBCs is also responsive to a PKR activator, and the resulting decrease in 2,3-DPG increases HbS-O₂ affinity. The black and green curves represent healthy donors and the blue and dashed-red curves represent SCD donors. Reduction in p50 indicates an increase in hemoglobin affinity for oxygen. As illustrated in FIG. 14 , Compound 1 normalizes the SCD oxygen affinity, resulting in overlap of the dashed-red Compound 1-treated SCD donor curve with the black, untreated healthy donor curve.

Example 7: Reduction of the Point of Sickling in SCD RBCs

The biologic consequences of increased PKR activation by Compound 1 in sickle RBCs is demonstrated in FIG. 15 . We observed an effect of Compound 1 on SCD RBC sickling was measured by the deformability or elongation index, or EI, of the sickle RBC under decreasing (and then increasing) levels of oxygen and the Point of Sickling, or POS, defined as the pO2 concentration where a decrease in EI is observed. As shown in FIG. 15 , comparison of the solid and dashed curves measuring pO2 concentration in the presence and absence of Compound 1, respectively, demonstrates that Compound 1 treatment improves RBC deformability at a lower oxygen tension suggesting that the Compound 1 treated sickle RBC can maintain a higher level of deformability as the RBCs transverse the microvasculature at lower oxygen levels.

FIGS. 16 and 17 provide further data demonstrating that Compound 1 improves deformability under de-oxygenation in vitro in HbS RBCs. As shown in FIG. 16 , HbS RBCs treated with Compound 1 in vitro had a lower P50 than HbS RBCs treated with DMSO. As shown in FIG. 17 , HbS RBCs treated with Compound 1 (20 μM) had a greater elongation index than HbS RBCs treated with DMSO, as measured by oxygen scan (oxygen gradient ektacytometry).

Example 8: Pharmacokinetic/Pharmacodynamic Studies of Compound 1 in Wild Type Mice

Two pharmacokinetic (PK)/pharmacodynamic (PD) studies were conducted in Balb/c mice that were administered Compound 1 once daily by oral gavage (formulated in 10% Cremophor EL/10% PG/80% DI water) for 7 days (QD×7) at doses of 0 (vehicle), 3.75, 7.5, 15, 30, 60 mg/kg (Study 1); 0 (vehicle), 7.5, 15, 30, 60, 120, or 240 mg/kg (Study 2). On the 7th day, whole blood was collected 24 hours after dosing and snap frozen. Samples were later thawed and analyzed by LC/MS for 2,3-DPG and ATP levels. In both studies, Compound 1 was well tolerated. No adverse clinical signs were observed and there were no differences in body weight change compared with the vehicle group.

The levels of 2,3-DPG decreased with Compound 1 treatment (FIGS. 18A and 18B (Studies 1 and 2) and FIG. 19 (Study 2)). In general, reductions were >20% at ≥15 mg/kg Compound 1, and >30% for 120 and 240 mg/kg Compound 1. Together, the results from the highest doses provide in vivo evidence that 2,3-DPG decreases with PKR activation.

Evaluation of ATP levels in these studies showed that treatment with Compound 1 increased levels of ATP. In Study 1, ATP increased 21% and 79% with 30 and 60 mg/kg Compound 1, respectively, compared to vehicle, and in Study 2, ATP levels increased with exposure with doses up to 120 mg/kg Compound 1 with a maximum increase of ˜110% compared to vehicle (FIG. 20A and FIG. 20B). At the highest dose, 240 mg/kg Compound 1, ATP levels increased by 45%. Levels of ATP correlated with Compound 1 exposure in a manner similar across both studies.

Example 9: Compound 1 Increases ATP Concentrations and Reduces 2,3-DPG in a Dose-Dependent Manner in Non-Human Primates

ATP levels in blood cells from non-human primates receiving daily doses of Compound 1 at 100, 300 and 550 mg/kg for 28 days were measured. Dose dependent increases in ATP levels were observed, reaching 90% at the highest dose of 550 mg/kg compared to pre-treatment, as illustrated in FIG. 21 .

Example 10: Compound 1 Reduces 2,3-DPG Concentrations in a Dose-Dependent Manner in Non-Human Primates

2,3-DPG levels in blood cells from non-human primates receiving daily doses of Compound 1 at 100, 350 and 550 mg/kg for 28 days were measured. Dose dependent decreases in 2,3-DPG levels were observed, with up to a 40% decrease from pretreatment levels, as illustrated in FIG. 22 .

Example 11: Testing a PKR Activating Compound in a Berkeley SS Mouse Model

Targeted gene deletions and a human transgene in the Berkeley SS mouse model result in mice that express human HgbS almost exclusively (Pászty C. Transgenic and gene knock-out mouse models of sickle cell anemia and the thalassemias. Curr Opin Hematol. 1997 March; 4(2):88-93) and mimic the genetic, hematologic, and histopathologic features of SCD in humans. These include irreversibly sickled RBCs, anemia, and multi-organ pathology.

The effects of Compound 1 on Hgb affinity for oxygen, percentage of sickled cells, and/or Hgb were evaluated in the Berkley SS mouse. Two studies were conducted where mice received 0 or 1000 ppm Compound 1 ad libitum in mouse chow for 7 days. Blood was drawn on Day 7 and analyzed under hypoxic conditions. Red cell parameters were measured by ADVIA.

The findings were relatively consistent between the two studies. Oxygen affinity to Hgb under hypoxic conditions was increased and p50 was decreased (FIG. 23A, FIG. 23B, FIG. 24A, and FIG. 24B). A positive effect on sickling was observed such that the percentage of RBCs that sickled at low oxygen pressures was decreased (FIG. 25 ) and time to sickle was increased with Compound 1 treatment. Furthermore, Hgb was increased by ≈1 g/dL. Together, these changes support the hypothesis that Compound 1 will reduce cell sickling and positively impact the downstream clinical complications of sickled RBCs. Compound 1 also reduced the level of reticulocytes in treated mice (FIG. 26 ), indicative of reduced red cell lysis.

Example 12: A SAD/MAD Study to Assess the Safety, Pharmacokinetics, and Pharmacodynamics of Compound 1 in Healthy Volunteers and Sickle Cell Disease Patients

Compound 1 is evaluated in a randomized, placebo-controlled, double blind, single ascending and multiple ascending dose study to assess the safety, pharmacokinetics, and pharmacodynamics of Compound 1 in healthy volunteers and sickle cell disease patients. The use of Compound 1 is disclosed herein for treatment of sickle cell disease in humans.

The hallmark of sickle cell disease (SCD) is hemoglobin S (HbS) polymerization upon deoxygenation, resulting in red blood cell (RBC) sickling and subsequent oxidative/membrane damage, hemolysis, inflammation, cell adhesion, and vasoocclusions. Exacerbating the pathogenesis of SCD, the HbS RBC has 1) increased 2,3-DPG with decreased oxygen affinity (increased p50) (see FIG. 27 ); and 2) decreased RBC ATP. Indeed, sickle RBCs contain more 2,3-DPG than healthy RBCs, resulting in decreased hemoglobin O₂ affinity (i.e., increased p50) and early release of O₂ (leading to deoxygenation of HbS, polymerization, and sickling). Sickle RBCs also have insufficient energy (i.e., less ATP than normal RBCs) for membrane maintenance and repair, contributing to hemolysis and reduced RBC lifespan.

Compound 1 is a novel, small molecule allosteric activator of erythrocyte pyruvate kinase (PKR) and functions as an RBC metabolic modulator causing decreased 2,3-DPG and increased ATP levels in RBC. Compound 1 is an oral activator of the Pyruvate Kinase R (PKR) that decreases 2,3-DPG and increases ATP in erythrocytes. As shown in FIG. 4 , (1) the reduction in 2,3-DPG may result in an increase in O₂ affinity of HbS, thereby reducing HbS polymerization and RBC sickling; and (2) the increase in ATP production may improve sickle RBC repair and membrane health, reducing hemolysis. Thus, the multimodal action of Compound 1 may improve hemoglobin levels and reduce the rate of vaso-occlusion in patients with SCD. In preclinical safety studies, Compound 1 had no effect on steroidogenesis, demonstrated low risk of drug-to-drug interactions, and was well tolerated in vivo at the maximum doses administered. A first-in-human Phase 1 study evaluating Compound 1 in healthy subjects (HS) and subjects with SCD has been initiated. The aims of this study are to evaluate the safety and PK/PD of Compound 1 in HS and subjects with SCD.

As illustrated in FIG. 28 , the trial to assess the safety and PK/PD of Compound 1 is a randomized, placebo-controlled, double blind, single dose and MAD trial in healthy adult volunteers and a single dose and MAD trial in adolescent or adult patients with SCD. The trial also includes a 12-week dosing cohort in which up to 20 SCD patients will each receive up to 84 consecutive daily doses of Compound 1.

Compound 1 is an oral small-molecule agonist of pyruvate kinase red blood cell isozyme (PKR) being developed for the treatment of hemolytic anemias. This human clinical trial study will characterize the safety, tolerability and the pharmacokinetics/pharmacodynamics (PK/PD) of a single ascending dose and multiple ascending doses of Compound 1 in the context of phase 1 studies in healthy volunteers and sickle cell disease patients. The effects of food on the absorption of Compound 1 will also be evaluated, in healthy volunteers.

The objectives of the study include the following:

-   -   1. To evaluate the safety and tolerability of a single ascending         dose and multiple ascending doses of Compound 1 in healthy         volunteers and sickle cell disease (SCD) patients.     -   2. To characterize the pharmacokinetics (PK) of Compound 1.     -   3. To evaluate the levels of 2,3-diphosphoglycerate (DPG) and         adenosine triphosphate (ATP) in the red blood cells (RBCs) of         healthy volunteers and SCD patients after single and multiple         doses of Compound 1.     -   4. To evaluate the relationship between Compound 1 plasma         concentration and potential effects on the QT interval in         healthy volunteers.     -   5. To evaluate the effect of single ascending doses of Compound         1 on other electrocardiogram (ECG) parameters (heart rate, PR         and QRS interval and T-wave morphology) in healthy volunteers.     -   6. To explore food effects on the PK of Compound 1 in healthy         volunteers.     -   7. To explore the association of Compound 1 exposure and         response variables (such as safety, pharmacodynamics (PD),         hematologic parameters as appropriate).     -   8. To explore effects of Compound 1 after single and multiple         doses on RBC function.     -   9. To explore effects of Compound 1 after multiple doses in SCD         patients on RBC metabolism, inflammation and coagulation.     -   10. To explore effects of Compound 1 on RBC hemoglobin-O₂         affinity and membrane mechanics.

This is a first-in-human (FIH), Phase 1 study of Compound 1 that will characterize the safety, PK, and PD of Compound 1 after a single dose and after repeated dosing first in healthy adult volunteers and then in adolescents or adults with sickle cell disease. The study arms and assigned interventions to be employed in the study are summarized in Table 10. Initially, a dose range of Compound 1 in single ascending dose (SAD) escalation cohorts will be explored in healthy subjects. Enrollment of healthy subjects into 2-week multiple ascending dose (MAD) escalation cohorts will be initiated once the safety and PK from at least two SAD cohorts is available to inform the doses for the 2-week MAD portion of the study. The MAD cohorts will then run in parallel to the single dose cohorts. A single dose cohort is planned to understand food effects (FE) on the PK of Compound 1. After the SAD and FE studies in healthy subjects are completed, the safety, PK and PD of a single dose of Compound 1 that was found to be safe in healthy subjects will then be evaluated in sickle cell disease (SCD) subjects. Multiple dose studies in SCD subjects will then be initiated upon completion of MAD studies in healthy volunteers. Compound 1 will be administered in 25 mg and 100 mg tablets delivered orally.

In this study, SAD/MAD cohorts are randomized (3 to 1) to receive Compound 1 or placebo (P). Compound 1 was evaluated first in 4 healthy SAD cohorts and 4 healthy MAD (14-day dosing period) cohorts. Based on the safety, and PK/PD profile from HS, Compound 1 is then evaluated in 1 SCD SAD cohort and 2 SCD MAD cohorts. Specifically, based on the safety and pharmacokinetic/pharmacodynamics (PK/PD) profile in healthy volunteer studies, Compound 1 is evaluated in patients (pts) with SCD, first in a single dose (SD or SAD) cohort and then in multiple-dose (MD or MAD) cohorts (14-day and 12-week). Safety assessments include AEs, vital signs, ECGs and laboratory parameters. PK/PD blood sampling was performed on Day 1 (SAD/MAD) and Day 14 (MAD) and up to 72 h after the last dose and at the end-of-study visit. PD parameters included 2,3-DPG, ATP, and p50 in all cohorts with additional PD studies (including oxygen scan) performed only in the SCD cohorts. PD parameters included 2,3-DPG, ATP, p50, RBC deformability with controlled deoxygenation and reoxygenation (Lorrca® oxygen scan) and varying osmolality (Lorrca® osmoscan)). To maintain study blind, pt identifiers were removed when needed.

TABLE 10 Assigned Arms Interventions Experimental: Single ascending dose Drug: Compound cohorts in healthy subjects 1/Placebo Healthy volunteer subject cohorts Healthy volunteer randomized 6:2 receiving a single dose of subjects will receive Compound 1 or placebo. The first cohort Compound 1/ will receive 200 mg of Compound 1 or placebo and be placebo. Dose escalation will occur if monitored for side Compound 1 or placebo is tolerated. The effects while maximum dose of Compound 1 or undergoing placebo will be 1500 mg. Planned doses pharmacokinetics for the SAD cohorts are listed in Table 11. and pharmacodynamic studies Experimental: Multiple ascending dose Drug: Compound cohorts in healthy subjects 1/Placebo Healthy volunteer subject cohorts Healthy volunteer randomized 9:3 to receive Compound 1 or subjects will receive placebo for 14 days continuous dosing. Compound 1/ The first cohort will receive 100 mg of placebo and be Compound 1 or placebo daily × 14 days. monitored for Alternatively, the first cohort will receive side effects 200 mg (e.g., 100 mg BID or 200 mg QD) while undergoing of Compound 1 or placebo daily × 14 pharmacokinetics days. The maximum dose of Compound and 1/placebo will be 600 mg Compound pharmacodynamic 1/placebo daily for 14 days. Planned studies doses for the MAD cohorts are listed in Table 12. Experimental: Food Effect Cohort in Drug: Compound 1 healthy subjects Healthy subjects Healthy Volunteer subject cohort of 10 will receive subjects who will receive a single dose of Compound 1 Compound 1 with food and without food. with or without Dose will be administered per the protocol food and undergo defined dose. Healthy Volunteer subject pharmacokinetic cohort of 10 subjects who will receive a studies single dose of Compound 1 with food and without food. Dose will be 500 mg of Compound 1, but is subject to change based on the pharmacokinetic profile of Compound 1 observed in the initial SAD cohorts and the safety profile of Compound 1 observed in prior SAD and MAD cohorts. Experimental: Single ascending dose Drug: Compound cohorts in SCD subjects 1/Placebo Sickle cell disease subject cohort SCD subjects randomized 6:2 receiving a single dose of will receive Compound 1 or placebo. The dose of Compound 1/ Compound 1/placebo administered will be placebo a dose that was found to be safe in healthy and be monitored subjects. The dose of Compound for side effects 1/placebo administered also will be a dose while undergoing that was found to be pharmacokinetic pharmacodynamically active (e.g., results and in a reduction in 2,3-DPG) in healthy pharmacodynamics subjects. studies One single dose cohort in SCD patients is planned to evaluate the safety and PK/PD of Compound 1 within the dose range of Compound 1 previously demonstrated to be tolerable in the healthy subject SAD cohorts, with a minimum of eight SCD patients to be randomly assigned to receive one dose of Compound 1 700 mg (n = 6) or 1 dose of placebo (n = 2). Experimental: Multiple ascending dose Drug: Compound 1/ cohorts in SCD subjects Placebo Sickle cell disease SCD subjects will subject cohorts randomized receive Compound 9:3 to receive Compound 1 or 1/placebo and be placebo for 14 days continuous dosing. monitored for side The dose of Compound 1/placebo effects while administered will be a dose less than undergoing maximum tolerable dose evaluated in pharmacokinetic MAD healthy volunteers. The dose of and Compound 1/placebo also will be a dose pharmacodynamics that was found to be studies pharmacodynamically active (e.g., results in a reduction in RBC 2,3-DPG and increase in RBC ATP) in MAD healthy volunteers. Up to two MAD cohorts in SCD patients are planned, with 12 patients per cohort to be screened, enrolled and randomly assigned to receive 14 consecutive daily doses of Compound 1 (n = 9) or placebo (n = 3). The initial daily dose of Compound 1 300 mg for 14 days to be evaluated in SCD patients was selected from the daily dose range of Compound 1 evaluated in the healthy adult volunteers that was found to be tolerable and pharmacodynamically active. If the safety results of the first MAD dose are acceptable and the PK/PD data are supportive, patients may be dosed with an additional daily dose of Compound 1 for 14 days. Experimental: 12-week dosing cohort Drug: Compound 1 in SCD subjects SCD subjects will Sickle cell disease receive Compound subjects cohort (n = up to 20) to 1 and be monitored receive up to 84 consecutive daily doses for side effects while of open-label Compound 1. The dose undergoing of Compound 1 administered with not pharmacokinetics exceed the highest dose evaluated in the and MAD SCD subject cohorts. pharmacodynamics studies

TABLE 11 Dose Level/Cohort Dose Tablet Strength (#/day) SAD 1 200 mg 100 mg (2/day) SAD 2 400 mg 100 mg (4/day) SAD 3 700 mg 100 mg (7/day) SAD 4 1100 mg  100 mg (11/day) SAD 5 1500 mg  100 mg (15/day)

TABLE 12 Dose Level/Cohort Dose Tablet Strength (#/day) MAD 1 100 mg 100 mg (1/day) or 25 mg (4/day) MAD 2 200 mg 100 mg (2/day) MAD 3 400 mg 100 mg (4/day) MAD 4 600 mg 100 mg (6/day)

Outcome Measures

Primary Outcome Measures:

1. Incidence, frequency, and severity of adverse events (AEs) per CTCAE v5.0 of a single ascending dose and multiple ascending doses of Compound 1 in adult healthy volunteers and SCD patients.

[Time Frame: Up to 3 weeks of monitoring]

2. Maximum observed plasma concentration (Cmax)

[Time Frame: Up to 3 weeks of testing]

3. Time to maximum observed plasma concentration (Tmax)

[Time Frame: Up to 3 weeks of testing]

4. Area under the plasma concentration-time curve from time zero until the 24-hour time point (AUC0-24)

[Time Frame: Up to 3 weeks of testing]

5. Area under the plasma concentration-time curve from time zero until last quantifiable time point (AUC0-last)

[Time Frame: Up to 3 weeks of testing]

6. Area under the plasma concentration-time curve from time zero to infinity (AUC0-inf)

[Time Frame: Up to 3 weeks of testing]

7. Terminal elimination half-life (t½)

[Time Frame: Up to 3 weeks of testing]

8. Apparent clearance (CL/F)

[Time Frame: Up to 3 weeks of testing]

9. Apparent volume of distribution (Vd/F)

[Time Frame: Up to 3 weeks of testing]

10. Terminal disposition rate constant (Lz)

[Time Frame: Up to 3 weeks of testing]

11. Renal clearance (CIR)

[Time Frame: Up to 3 weeks of testing]

Secondary Outcome Measures:

12. Change from baseline in the levels of 2,3-diphosphoglycerate (DPG) and adenosine triphosphate (ATP) in the red blood cells (RBCs) of healthy volunteers and SCD patients after single and multiple doses of Compound 1.

[Time Frame: Up to 3 weeks of testing]

13. Model-based estimate of change from baseline QT interval corrected using Fridericia's correction formula (QTcF) and 90% confidence interval at the estimated Cmax after a single dose of Compound 1 in healthy volunteers.

[Time Frame: up to 7 days]

14. Change from baseline heart rate after a single dose of Compound 1 in healthy volunteers

[Time Frame: up to 7 days]

15. Change from baseline PR after a single dose of Compound 1 in healthy volunteers

[Time Frame: up to 7 days]

16. Change from baseline QRS after a single dose of Compound 1 in healthy volunteers

[Time Frame: up to 7 days]

17. Change from baseline T-wave morphology after a single dose of Compound 1 in healthy volunteers

[Time Frame: up to 7 days]

Exploratory Outcome Measures:

18. Effect of food on Cmax, AUC₀₋₂₄/AUC_(last) 19. Effect of AUC_(last)/AUC₀₋₂₄, Cmax, minimum plasma concentration (C_(min)), peak-to trough ratio, dose linearity, accumulation ratio on safety, PD, and hematologic parameters of interest, as assessed by exposure-response analyses 20. Effect of 2,3-DPG reduction in RBCs on the oxyhemoglobin dissociation curve (p50; partial pressure of O₂ at which 50% of hemoglobin is saturated with O₂) after a single dose and after chronic dosing of Compound 1 21. Effect of chronic Compound 1 dosing on normal and SCD RBC deformability by osmotic gradient ektacytometry and oxygen gradient ektacytometry 22. Effect of chronic Compound 1 dosing on SCD RBC response to oxidative stress in SCD Patients (including evaluation of glutathione, glutathione peroxidase and superoxide dismutase levels) 23. Effect of chronic Compound 1 dosing on measurable markers of inflammation in SCD Patients (C-reactive protein, ferritin, interleukin [IL]-1β, IL-6, IL-8, and tumor necrosis factor-α) 24. Effects of chronic Compound 1 dosing on measurable markers of hypercoagulation in SCD patients (D-dimer, prothrombin 1.2, and thrombin-antithrombin [TAT] complexes)

Eligibility

-   -   Minimum age: 18 Years (healthy volunteers); 12 Years (SCD         subjects)     -   Maximum age: 60 or 65 Years     -   Sex: All     -   Gender Based: No     -   Accepts Healthy Volunteers: Yes

Inclusion Criteria:

-   -   Healthy volunteer: subjects must be between 18 and 60 years of         age; SCD: subjects must be between 12 and 50 or 65 years of age     -   Subjects must have the ability to understand and sign written         informed consent, which must be obtained prior to any         study-related procedures being completed.     -   Healthy volunteer: Subjects must be in general good health,         based upon the results of medical history, a physical         examination, vital signs, laboratory profile, and a 12-lead ECG;         SCD: Previously diagnosed sickle cell disease (hemoglobin         electrophoresis or genotype).     -   Subjects must have a body mass index (BMI) within the range of         18 kg/m2 to 33 kg/m² (inclusive) and a minimum body weight of 50         kg (healthy volunteer subjects) or 40 kg (SCD subjects)     -   For SCD subjects, sickle cell disease previously confirmed by         hemoglobin electrophoresis or genotyping indicating one of the         following hemoglobin genotypes: Hgb SS, Hgb SR-thalassemia, Hgb         Sβ⁰-thalassemia, or Hgb SC     -   All males and females of child bearing potential must agree to         use medically accepted contraceptive regimen during study         participation and for 90 days after last study drug         administration.     -   Subjects must be willing to abide by all study requirements and         restrictions.

Exclusion Criteria (Healthy Volunteers):

-   -   Evidence of clinically significant medical condition or other         condition that might significantly interfere with the         absorption, distribution, metabolism, or excretion of study         drug, or place the subject at an unacceptable risk as a         participant in this study     -   History of clinically significant cardiac diseases including         condition disturbances     -   Abnormal hematologic, renal and liver function studies     -   History of drug or alcohol abuse

Exclusion Criteria (SCD Subjects):

-   -   Had more than 6 episodes of vaso-occlusive crisis (VOC) within         the past 12 months that required a hospital, emergency room, or         clinic visit     -   Had at least one episode of acute chest syndrome in the last 6         months     -   Received any of the following approved therapies for use in SCD:         -   Hydroxyurea (HU): excluded if started HU <90 days prior to             Day 1 of study treatment         -   crizanlizumab: excluded if received an infusion within 14             days prior to Day 1 of study treatment         -   voxelotor: excluded if received a dose within 7 days prior             to start of Day 1 of study treatment     -   Received a red blood cell transfusion within 30 days of starting         the study drug     -   Hemoglobin <7.0 g/dL or >10.5 g/dL     -   Unable to take and absorb oral medications

Results (Healthy Subjects)

At least 90 healthy volunteers have received Compound 1 (n=70) or placebo (n=20) in the Phase 1 trial. Eight SCD patients have received blinded trial drug or placebo as part of the single dose trial cohort (n=7) or as part of the first 14-day dose MAD 1) cohort (n=1). To date, Compound 1 has demonstrated a promising tolerability profile and time independent PK profile.

Compound 1 has been evaluated in the HS SAD/MAD/Food Effect cohorts (n=90) and in the SCD SAD cohort (n=6). In HS studies, Compound 1 was well tolerated and exhibited a favorable safety profile, with Grade 1 headache as the most common AE reported in HS receiving a single dose (4%) or 14 days (28%) of Compound 1 and in ⅙ SCD subjects receiving Compound 1/P (blinded). The PK profile of Compound 1 was similar in HS and SCD subjects. Compound 1 was rapidly absorbed with a median Tmax of 1 h postdose, a T½ of ˜10-13 h, and an AUC0-24 ˜7000 h·ng/mL. No effect on testosterone or estradiol levels was observed in healthy subjects.

In the HS studies, Compound 1 exhibited linear and time-independent PK, and the PD activity of Compound 1 was observed at all dose levels after 24 h (decreased 2,3-DPG, p<0.0001) and after 14-days (increased ATP, p<0.0001) of dosing. The biologic consequence of this PD response was an increase in oxygen affinity (decreased p50, p<0.0001) within 24 h of Compound 1 dosing and a decrease in absolute reticulocyte counts (p<0.0001) with a slight increase in hemoglobin levels (ns) by Day 4 of the dosing period in all Compound 1 dose cohorts.

Four healthy SAD cohorts were evaluated at doses of 200, 400, 700, and 1000 mg, and four healthy MAD cohorts received 200 to 600 mg total daily doses for 14 days at QD or BID dosing (100 mg BID, 200 mg BID, 300 mg BID, and 400 mg QD). In the food effect (FE) cohort, 10 healthy subjects received 200 mg of Compound 1 QD with and without food.

Demographics and baseline characteristics of the healthy volunteers in the SAD and MAD cohorts are provided in Table 13.

TABLE 13 Demographics and Baseline Characteristics SAD SAD MAD MAD Placebo Compound Placebo Compound Characteristic N = 8 1 N = 24 N = 12 1 N = 36 Age, years, 41 (6)    45 (11)  45 (12) 45 (11) (mean, SD) Male, n (%) 6 (75)  14 (58)  6 (50) 22 (61) Race, n (%) White 6 (75)  10 (42)  5 (42) 20 (56) Black 2 (25)  14 (58)  4 (33) 13 (36) Other/Multiple 0 0  3 (25) 3 (8) Weight, kg, 79 (15)   81 (14)  73 (13) 80 (9)  mean (SD) Height, cm, 171 (8)    173 (9)  170 (10) 173 (9)  mean (SD) BMI, kg/m², 27 (3)  27 (4) 25 (4) 27 (3)  mean (SD)

No serious adverse events (SAEs) or AEs leading to withdrawal were reported in the SAD and MAD cohorts of healthy volunteers. The treatment emergent adverse events recorded in the healthy volunteer cohorts are provided in Table 14. Among the TEAEs reported in Table 14, TEAEs of grade 2 or less related to Compound 1 in the SAD cohorts included headache (n=1) and transient ventricular tachycardia (n=1), each in a different subject. TEAEs of grade 2 or less related to Compound 1 in the MAD cohorts included headache (n=4), palpitations (n=1) and somnolence (n=1), each in a different subject. TEAEs of grade 2 or less in the placebo cohorts included headache in one subject. One grade 3 TEAE unrelated to Compound 1. Transient asymptomatic lipase elevation was noted in one subject at the 1000 mg dose. The subject's back-up sample was re-assessed independently, and no lipase elevation was detected.

TABLE 14 Healthy Volunteers: Treatment Emergent Adverse Events SAD SAD MAD MAD Placebo Compound Placebo Compound Characteristic N = 8 1 N = 24 N = 12 1 N = 36 Any TEAE, n (%) 1 (13) 5 (21) 3 (25) 15 (42) Any grade 3 or 0 1 (4)  0 0 greater TEAE, n (%) Drug interruption, 0 0 0 0 reduction, or discontinuation due to TEAE, n (%)

In PK assessments, Compound 1 was rapidly absorbed with a median Tmax of 1 hr postdose. FIG. 29 illustrates plasma Compound 1 pharmacokinetics in healthy volunteers following a single dose. Linear pharmacokinetics was observed from single doses up to 700 mg, with a T_(1/2) of 11-15 hrs. Single dose exposure increased in greater than dose-proportional manner at doses ≥700 mg. In multiple-doses delivered BID or QD, linear PK was observed across all dose levels (100-300 mg BID, 400 mg QD), and exposure remained steady up to day 14, without cumulative effect. No significant changes in exposure were observed after 14 days of dosing. Compound 1 exposure under fed/fasted conditions was similar.

PD activity was demonstrated at all dose levels evaluated in Compound 1-treated subjects (Table 15). Table 15 reports the mean maximum percentage change in 2,3-DPG, ATP, and p50 across all doses and timepoints in the SAD and MAD cohorts. As shown in Table 15, a mean decrease in 2,3-DPG and p50, and a mean increase in ATP, relative to baseline, was observed in both the SAD and MAD cohorts. Within 24 hr of a single dose of Compound 1, a decrease in 2,3-DPG with a corresponding increase in p50 was observed. After 14 days of Compound 1 dosing these PD effects were maintained along with an increase in ATP over baseline. Accordingly, the mean maximum reduction in the concentration of 2,3-DPG was at least about 40% in patients receiving Compound 1 in the SAD study (range 35.4-56.1%) and at least about 50% in patients receiving Compound 1 in the MAD study (range 46.1-63.6%).

TABLE 15 Summary of Mean Maximum Percent Change in Key PD Measures from Baseline SAD MAD PD Placebo Compound Placebo Compound Marker Statistics (N = 8) 1 (N = 24) (N = 12 1 (N = 36) 2,3- Mean −19.5 −46.8 −17.0 −56.3 DPG (95% CI) (−25.0, (−50.3, (−22.9, (−58.9, −14.0) −43.2) −11.1) −53.7) P-value <0.0001 <0.0001 ATP Mean 9.2 24.4 7.2 68.5 (95% CI) (0.5, (18.4, (−0.3, (63.6, 18.0) 30.3) 14.7) 73.3) P-value 0.0094 <0.0001 p50 Mean 0.9 −15.6 -0.8 −15.9 (95% CI) (−1.2, (−17.5, (−3.0, (−17.2, 2.9) −13.8) 1.4) −14.5) P-value <0.0001 <0.0001

In the SAD cohorts, the subjects' blood 2,3-DPG levels were measured periodically after dosing by a qualified LC-MS/MS method for the quantitation of 2,3-DPG in blood. Decreased 2,3-DPG blood levels were observed 6 hours following a single dose of Compound 1 at all dose levels (earlier timepoints were not collected). Maximum decreases in 2,3-DPG levels generally occurred ˜24 hours after the first dose with the reduction sustained ˜48-72 hr postdose. Table 16 reports the median percentage change in 2,3-DPG blood levels, relative to baseline, measured over time in healthy volunteers after a single dose of Compound 1 (200 mg, 400 mg, 700 mg, or 1000 mg) or placebo. Accordingly, the median reduction in the concentration of 2,3-DPG, relative to baseline, was at least about 3000 at all dose levels tested 24 hours after administration of the single dose.

TABLE 16 Median Percentage Change in 2,3-DPG Levels Time After Dose Dose Placebo 200 mg 400 mg 700 mg 1000 mg 0 0.0 0.0 0.0 0.0 0.0 6 −7.8 −18 −23 −29 −20 8 −7.6 −17 −29 −28 −31 12 −4.0 −25 −40 −41 −44 16 −6.0 −33 −35 −46 −50 24 −2.0 −31 −39 −49 −48 36 −6.9 −33 −38 −46 −47 48 −15 −29 −31 −48 −47 72 −6.9 −18 −30 −33 −21

FIG. 30 is a graph of the blood 2,3-DPG levels measured over time in healthy volunteers who received a single dose of Compound 1 (200 mg, 400 mg, 700 mg, or 1000 mg) or placebo. As shown in FIG. 30 , healthy volunteers who received Compound 1 experienced a decrease in blood 2,3-DPG levels, relative subjects who received the placebo. FIG. 31A is a table of data obtained from the healthy subjects in a single ascending dose (SAD) clinical study of Compound 1 described in Example 12. As shown in FIG. 31A, dose normalized Cmax and AUC increased with increasing doses ≥700 mg suggesting greater than dose proportional increases in exposure at the highest doses tested. FIG. 31B is a table of data obtained from the healthy subjects in a multiple ascending dose (MAD) human clinical study of Compound 1 described in Example 12, showing time-independent pharmacokinetic (PK) properties over 14 days of dosing Compound 1 either QD or BID. In the tables of FIGS. 31A and 31B, AUC refers to the area under the concentration-time curve; BID refers to twice daily administration of Compound 1; C_(max) refers to the maximum concentration; QD refers to once daily administration of Compound 1; T_(max) refers to the time to maximum concentration of Compound 1. Values in FIG. 31B are presented as geometric mean [CV %] for C_(max), AUC_(0-tau), R Cmax, and R AUC_(0-tau); T_(max) presented as median [CV %].

FIG. 32 is a graph of the blood 2,3-DPG levels measured 24 hours post-dose in healthy volunteers who received a single dose of Compound 1 (200 mg, 400 mg, 700 mg, or 1000 mg) or placebo. As shown in FIG. 32 , healthy volunteers who received Compound 1 experienced a decrease in blood 2,3-DPG levels at 24 hours post-dose, relative to subjects who received the placebo.

In the SAD cohorts, the subjects' p50 (PO₂ at 50% hemoglobin saturation) were determined 24-hours post-dose. p50 measured 24 hours after a single dose of Compound 1 were reduced at all dose levels tested (median reduction ranged from ˜3-5 mmHg). Table 17 reports the mean absolute change in p50, relative to baseline, measured 24 hours after a single dose of Compound 1 (200 mg, 400 mg, 700 mg, or 1000 mg) or placebo in healthy volunteers.

TABLE 17 Mean Absolute Change in p50 (mmHg) Dose Mean Absolute Change Placebo 0.20 200 mg −2.91 400 mg −3.41 700 mg −4.85 1000 mg  −5.05

Following single doses, all HVs receiving Compound 1 exhibited a PD response associated with decreased p50 (increased Hb oxygen affinity). FIG. 33 is a graph of the p50 values measured 24 hours post-dose in healthy volunteers who received a single dose of Compound 1 (200 mg, 400 mg, 700 mg, or 1000 mg) or placebo. As shown in FIG. 33 , healthy volunteers who received Compound 1 experienced a decrease in p50, relative to subjects who received the placebo. FIG. 34 is a graph of the p50 values measured pre-dose and 24-hours post-dose in healthy volunteers who received a single dose of Compound 1 (200 mg, 400 mg, 700 mg, or 1000 mg) or placebo. As shown in FIG. 34 , healthy volunteers who received Compound 1 experienced a decrease in p50 relative to baseline, reflecting an increase in oxygen affinity, while subjects who received the placebo did not.

In the MAD cohorts, the subjects' blood 2,3-DPG levels were measured periodically after dosing by a qualified LC-MS/MS method for the quantitation of 2,3-DPG in blood. The maximum decrease in 2,3-DPG on Day 14 was 55% from baseline (median). 2,3-DPG levels reached a nadir and plateaued on Day 1 and had not returned to baseline levels 72 hours after the final dose on Day 14. Table 18 reports the median percentage change in 2,3-DPG blood levels, relative to baseline, measured over time after the first dose on days 1 and 14 in healthy volunteers who received daily doses of Compound 1 (100 mg BID, 200 mg BID, or 300 mg BID) or placebo for 14 days. Accordingly, the median reduction in the concentration of 2,3-DPG, relative to baseline, was at least about 25% at all dose levels tested 24 hours after administration of the first dose on day 1 and at least about 40% at all dose levels tested 24 hours after administration of the first dose on day 14.

TABLE 18 Median Percentage Change in 2,3-DPG Levels (Days 1 and 14) Dose 100 mg BID 200 mg BID 300 mg BID Placebo Time After Day Day Day Day First Daily Dose 1 14 1 14 1 14 1 14 0 0.0 −42.0 0.0 −48.2 0.0 −59.4 0.0 −7.6 6 −16.1 −44.3 −13.1 −48.5 −18.8 −53.0 −2.9 −10.9 8 −12.1 −44.7 −22.3 −44.3 −23.8 −54.2 −0.6 −1.6 12 −18.1 −43.6 −23.1 −42.2 −31.6 −55.3 −7.1 −1.6 16 −18.4 −43.9 −33.9 −42.9 −40.7 −52.4 −6.7 −5.3 24 −27.8 −44.1 −43.5 −44.3 −50.8 −52.1 1.1 −10.7 48 −34.7 −38.7 −44.5 −1.0 72 −20.2 −20.2 −32.9 −7.0

FIGS. 35 and 36 are graphs of the blood 2,3-DPG levels measured over time in healthy volunteers who received daily doses of Compound 1 (100 mg BID, 200 mg BID, 300 mg BID, or 400 mg QD) or placebo for 14 days. As shown in FIG. 35 , healthy volunteers who received Compound 1 experienced a decrease in blood 2,3-DPG levels, relative subjects who received the placebo. As illustrated in FIG. 36 , in RBCs of healthy volunteers, Compound 1 has demonstrated a reduction in 2,3-DPG, thus providing support for PKR activation in healthy RBCs. Notably, these effects were maintained for more than one day after Compound 1 dosing was stopped at day 14. PK/PD modelling predicts maximal 2,3-DPG response at doses ≥150 mg BID or ≥400 mg QD in HV RBCs. FIG. 37 is a graph of the blood 2,3-DPG levels measured on day 14 in healthy volunteers who received daily doses of Compound 1 (100 mg BID, 200 mg BID, 300 mg BID, or 400 mg QD) or placebo for 14 days. As shown in FIG. 37 , healthy volunteers who received Compound 1 experienced a decrease in blood 2,3-DPG levels, relative to subjects who received the placebo.

In the MAD cohorts, the subjects' p50 (PO₂ at 50% hemoglobin saturation) were determined on day 14. p50 values measured after 14 days of twice daily dosing were reduced at all dose levels tested (median reduction ranged from −3-5 mmHg). Table 19 reports the mean absolute change in p50, relative to baseline, measured on day 14 in healthy volunteers who received daily doses of Compound 1 (100 mg BID, 200 mg BID, or 300 mg BID) or placebo for 14 days.

TABLE 19 Mean Absolute Change in p50 (mmHg) (Day 14) Dose Mean Absolute Change Placebo −0.38 100 mg BID −3.26 200 mg BID −4.14 300 mg BID −5.34

Following multiple doses, all HVs receiving Compound 1 exhibited a PD response associated with decreased p50 (increased Hb oxygen affinity). FIG. 38 is a graph of the p50 values measured on day 14 in healthy volunteers who received daily doses of Compound 1 (100 mg BID, 200 mg BID, 300 mg BID, or 400 mg QD) or placebo for 14 days. As shown in FIG. 38 , healthy volunteers who received Compound 1 experienced a decrease in p50, relative to subjects who received the placebo. FIG. 39 is a graph of the p50 values measured pre-dose and on day 14 in healthy volunteers who received daily doses of Compound 1 (100 mg BID, 200 mg BID, 300 mg BID, or 400 mg QD) or placebo for 14 days. As shown in FIG. 39 , healthy volunteers who received Compound 1 experienced a decrease in p50 relative to baseline, reflecting an increase in oxygen affinity, while subjects who received the placebo did not.

In the MAD cohorts, the subjects' blood ATP levels were measured on day 14 by a qualified LC-MS/MS method for the quantitation of ATP in blood. ATP levels were elevated, relative to baseline, on day 14, and remained elevated 60 hours after the last dose. Table 20 reports the median percentage change in blood ATP levels, relative to baseline, measured over time after the first dose on day 14 in healthy volunteers who received daily doses of Compound 1 (100 mg BID, or 200 mg BID) or placebo for 14 days.

TABLE 20 Median Percentage Change in ATP Levels (Day 14) Time After First Dose Daily Dose 100 mg BID 200 mg BID Placebo 0 41.5 55.3 −0.5 6 43.8 48.1 2.8 8 47.8 58.4 −4.1 12 45.4 56.2 2.3 16 44.8 57.0 −6.8 24 55.0 64.0 2.9 48 52.2 58.9 4.7 72 49.2 54.0 2.2

FIG. 40 is a graph of the blood ATP levels measured on day 14 in healthy volunteers who received daily doses of Compound 1 (100 mg BID, 200 mg BID, 300 mg BID, or 400 mg QD) or placebo for 14 days. As shown in FIG. 40 , healthy volunteers who received Compound 1 experienced an increase in blood ATP levels, relative to subjects who received the placebo.

As illustrated in FIG. 41 , in RBCs of healthy volunteers, Compound 1 has demonstrated an increase in ATP, thus providing support for PKR activation in healthy RBCs. Notably, these effects were maintained for more than three days after Compound 1 dosing was stopped at day 14. PK/PD modelling predicts maximal ATP response at doses ≥50 mg BID or ≥150 mg QD in HV RBCs.

FIG. 42 is a graph showing the difference in the p50 values determined pre-dose and 24 hours post-dose (SAD cohorts) and 24 hours post-dose on day 14 (MAD cohorts) in healthy volunteers who received Compound 1 or placebo. As shown in FIG. 42 , healthy volunteers who received Compound 1 experienced a change (decrease) in p50 relative to baseline, while subjects who received the placebo did not.

FIG. 43 is a graph plotting the blood concentration of Compound 1 (ng/mL) measured in healthy volunteer (HV) patients on a first (left) axis and the concentration of 2,3-DPG (micrograms/mL) measured in these HV patients on a second (right) axis after administration of a single dose of Compound 1 (400 mg). Solid symbols represent geometric means and Standard errors of the observed Compound 1 plasma and 2,3 DPG concentrations. As shown in the figure, the observed 2,3 DPG modulation does not track directly plasma pharmacokinetics (blood concentration of Compound 1) where the pharmacodynamic maximum (i.e., the minimum of the 2,3-DPG concentration, at time ˜24 h) occurred nearly 24 h after the pharmacokinetic maximum (i.e., maximum of the PK curve, at time ˜1-2 h). The observed pharmacodynamic response in HVs was durable, where 2,3-DPG depression was observed long after plasma Cmax. Taken together, this suggests that identifying the pharmacologically active dose cannot be adequately performed using pharmacokinetic parameters (C_(max)/C_(min)/AUC) in isolation, but rather support an approach that includes integrating the temporal pharmacokinetic/pharmacodynamic relationship to provide the platform of evidence that QD dosing may be feasible in sickle cell disease patients.

FIG. 44 is a scatter plot of 2,3-DPG levels and p50 values observed in healthy volunteers in the SAD and MAD cohorts. Solid symbols represent the observed p50/2,3-DPG levels in healthy volunteers dosed with Compound 1 at 24 h following the last administered dose. Baseline data represents p50/2,3 DPB data obtained either prior to Compound 1 treatment and from healthy volunteers dosed with placebo. A positive correlative relationship between 2,3 DPG and p50 levels was observed for patients receiving various doses. As illustrated in FIG. 45 , the increase in oxygen affinity in subjects treated with Compound 1 correlated with the reduction of 2,3-DPG, demonstrating preliminary proof of mechanism in healthy RBCs and supporting further clinical development of Compound 1 in patients with SCD.

Results (SCD Subjects)

Modeling of pharmacodynamic response in healthy volunteer RBCs indicated that doses of Compound 1≥150 mg per day result in the maximum ATP response, and ≥400 mg per day maximize the 2,3-DPG response (FIG. 46 ). A potential exposure to the maximum PD response dose range to evaluate in patients with SCD was identified. Based on the safety and PK/PD profile in healthy volunteer studies, a 700 mg single dose was evaluated in patients with SCD (n=7). A single 700 mg dose of Compound 1 was selected to evaluate in patients with SCD to enable daily dosing cohorts at lower exposures.

The baseline characteristics of the SCD patients receiving a single 700 mg dose of Compound 1 or placebo are reported in Tables 21 and 22. All patients had a Hb SS genotype and a mild VOC history but persistent anemia and ongoing hemolysis, despite hydroxyurea therapy.

TABLE 21 Baseline Characteristics of SCD Patients Enrolled in Single Dose Cohort (N = 7) Age, years 34.7 (15, 48) Male  2 (29%) Hb SS genotype  7 (100%) Hydroxyurea therapy  7 (100%) 12-mo VOC rate 0 (0, 2) Prior packed RBC transfusion  1 (14%) (>30 days) Hemoglobin electrophoresis % HbS 79.6 (69.1, 88.6) % HbF 15.6 (7.9, 28.6)

TABLE 22 Baseline Characteristics of SCD Patients Enrolled in Single Dose Cohort (N = 7) Hb, g/dL 8.7 (7.1, 10.3) RBC, 10¹²/L 2.4 (1.8, 2.9)  ARC, 10⁹/L 196.0 (54.9, 350.3) Total bilirubin, mg/dL 3.54 (2.0, 6.2)    LDH, U/L  393.4 (317.0, 559.5) 2,3-DPG, μg/gHb 5291 (4602, 6137) ATP, μg/gHb 1845 (1552, 2158) p50, pO₂ mmHg 30.1 (26.1, 34.0) 

No serious adverse events (SAEs) or TEAEs leading to pt withdrawal were reported in the SD cohort. In the SD cohort, 7 pts (2 males, 5 females, all HbSS) received 700 mg Compound 1 (n=5) or placebo (n=2).

All SCD patients who received a single 700 mg dose of Compound 1 or placebo were monitored for adverse events for 7 days. The incidence of treatment emergent adverse events (TEAEs) in SCD patients receiving Compound 1 (700 mg) or placebo are reported in Table 23. Six TEAEs were reported in 4 patients; all TEAEs were grade 1 and transient. Specifically, six TEAEs were reported in 4 of 7 (570%) patients, including 3 TEAEs (arthralgia, headache, palpitations) in 2 of 5 (400%) pts receiving Compound 1 and 3 TEAEs (back pain, myalgia, pruritis) in 2 of 2 (100%) pts receiving placebo; all TEAEs were grade 1 and transient. In the Compound 1 cohort, arthralgia, headache, and palpitations each were observed in one patient. One possibly related TEAE (palpitations) occurred about 8 hours post dose. No other symptoms were observed, and the palpitations resolved in <1 minute. In the placebo cohort, back pain, myalgia, and pruritus each were observed in one patient. By comparison, no TEAEs were observed in healthy volunteers who received a single dose of Compound 1 (700 mg) or placebo. The single 700 mg dose of Compound 1 was considered tolerable, and the first multiple dose SCD cohort (“Dose 1” in FIG. 28 ) was initiated.

TABLE 23 Compound 1 is Well Tolerated in Patients with SCD Compound 1 Placebo 700 mg (N = 5) (N = 2) Any TEAE, n (%) 2 (40) 2 (100) Related to study drug, 1 (20) 0 n (%)

In 3 pts with SCD (3 females, all HbSS) who thus far completed MD-1, 14 days of 300 mg Compound 1 or placebo daily was well tolerated, with 1 pt reporting transient, unrelated Grade 2 TEAEs of nausea/vomiting at the end of the 14-day dosing period.

As shown in FIG. 47 and Table 24, similar Compound 1 plasma pharmacokinetic profiles were observed in healthy volunteers and SCD patients who received a single 700 mg dose of Compound 1.

TABLE 24 Plasma PK Parameters in Healthy Volunteers and Patients with SCD Single Dose C_(max) AUC_(inf) t_(1/2) T_(max) (700 mg) ng/mL (h · ng/mL) (h) (h) HV 2204 (83.5) 6995 (30.3) 13.3 (34.3) 0.5 (0.5, 6.0) (N = 6) SCD 2585 (59.9) 7300 (43.4) 14.9 (48.7) 2.0 (1.0, 4.0) (N = 5) Values are geometric mean (geometric coefficient of variation) except for T_(max) (Median [Min, Max]).

Biologic activity has been observed in SCD subjects receiving a single dose of Compound 1, demonstrating the PKR enzyme in the SCD RBC is functional and responds to an allosteric PKR activator. As shown in FIG. 48 , 24 hours after a single 700-mg dose of Compound 1 in patients with SCD, ATP blood concentrations increased by 30%, and 2,3-DPG blood concentrations decreased by 26%.

Increased O₂ affinity (↓ P₅₀) with a decreased point of sickling (PoS) and improved HbS RBC deformability were observed in all Compound 1-treated pts. Improved HbS RBC membrane function was also demonstrated with a shift of the osmoscan results towards normal. Improved hematologic parameters, including ˜0.9 g/dL Hb increase compared with placebo, were also observed 24 h after a single dose of Compound 1.

As shown in FIG. 49 , increased hemoglobin O₂ affinity (decreased p50) was observed after a single 700 mg dose of Compound 1 in both healthy volunteers (see also FIG. 34 ) and patients with SCD.

As shown in FIG. 50 , increased hemoglobin O₂ affinity correlated with a reduction in 2,3-DPG in both healthy volunteers (see also FIG. 40 ) and patients with SCD.

As shown in FIG. 51 , SCD patients treated with Compound 1 demonstrated improved hematologic parameters 24 hours after Compound 1, when maximum 2,3-DPG and ATP responses were observed (see FIG. 48 ), returning to baseline after 72 hours. A single dose of Compound 1 resulted in an increase in Hb of 0.5 g/dL (range: 0.3, 0.9) in Compound 1-treated participants vs. a decrease in Hb of 0.4 g/dL (range: −0.5, −0.3) in placebo-treated participants (decreased Hb potentially due to phlebotomy). Decreased lactate dehydrogenase (LDH) was also observed in Compound 1-treated participants 72 hours after Compound 1 dosing, indicating a reduction in RBC turnover as the source for the transient improvement in RBC parameters. These results suggest that a sustained 2,3-DPG and ATP response may be required for optimal benefit.

The effects of a single dose of Compound 1 (700 mg) versus placebo on oxygen scan, oxygen affinity (p50), and osmoscan in SCD patients were evaluated. At the Point of Sickling (POS or PoS), polymerization of de-oxy HbS can affect the deformability of the RBCs and the elongation Index starts to decrease. The EImin refers to the lowest level of RBC deformability in the Oxygenscan. The lower the EImin the lower the deformability of the RBC. As shown in FIG. 52 and Table 25 (Oxygenscan), Compound 1 decreased the deoxygenation HbS polymerization rate and improved sickle RBC O₂-dependent deformability, as demonstrated by reductions in POS and increases in EI_(min). This effect was observed in all participants receiving Compound 1. As shown in FIG. 53 and Table 25 (Oxygen affinity curve), Compound 1 increased O₂ affinity (decreased p50) in all participants treated. As shown in FIG. 54 and Table 25 (Osmoscan), Compound 1 improved osmolality-dependent membrane function in sickle RBCs, as demonstrated by improvements (i.e., shifts toward normal) in O_(min) and O_(hyper). These effects were transient, returning to baseline 3 to 7 days after the single dose of Compound 1.

TABLE 25 Improvement in Deformability, Oxygen Affinity, and Osmotic Fragility in Sickle RBCs Under Deoxygenation and/ or Shear Stress After a Single Dose of Compound 1 (700 mg) Post-dose P Parameter Pre-dose (24 hours) Value POS  35.4 (27.3, 38.8)  24.0 (17.9, 31.8) .063 (Oxygenscan) EI_(min) 0.193 (0.16, 0.21) 0.296 (0.26, 0.38) .125 (Oxygenscan) EI_(max) 0.445 (0.41, 0.51) 0.451 (0.42, 0.52) .250 (Oxygenscan) p50 (Oxygen  29.4 (26.1, 32.3)  25.8 (23.3, 26.8) .063 affinity curve) EI_(max) 0.483 (0.46, 0.57) 0.478 (0.46, 0.57) .750 (Osmoscan) O_(min) 108 (105, 121) 117 (106, 124) .063 (Osmoscan) O_(hyper) 380 (371, 399) 400 (371, 412) .125 (Osmoscan) Values presented as median (range). P values based on the nonparametric Wilcoxon rank sum test for paired data.

FIGS. 55A and 55B show the effects of Compound 1 on a SCD subject's RBCs, 24 h after Compound 1 dosing. As shown in FIG. 55A, SCD subjects who received a single dose of Compound 1 experienced increased oxygen affinity of HbS, similar to HbA. As shown in FIG. 55B, subjects who received a single dose of Compound 1 experienced a left shift in the point of sickling (PoS) with an increase in the EImin.

No serious adverse events (SAEs) or TEAEs leading to pt withdrawal were reported in the MD cohort as of Jul. 17, 2020. In 3 pts with SCD (3 females, all HbSS) who thus far completed MD-1, 14 days of 300 mg Compound 1 or placebo daily was well tolerated, with 1 pt reporting transient, unrelated Grade 2 TEAEs of nausea/vomiting at the end of the 14-day dosing period.

Laboratory changes relative to pretreatment for each pt in the MD cohort as of Jul. 17, 2020 are shown in Table 26. In 2 of 3 SCD MD-1 pts treated with Compound 1/placebo (currently blinded), Hb increased by >1 g/dL, % reticulocytes decreased, and markers of hemolysis were improved after 14 days of treatment (compared to pre-treatment levels). Hematologic parameters returned to pre-treatment levels 4 to 7 days post-treatment (data not shown) without clinical AEs. Functional studies in the 2 pts with increased Hb showed improved RBC deformability (↓ PoS) and improved RBC membrane function while on study treatment relative to pre-treatment and/or post-treatment.

TABLE 26 Laboratory Changes in Patients with SCD Receiving 300 mg Compound 1/Placebo Once Daily for 14 Days Change Change from from Screen/Pre- Screen/Pre- Day 1 Day 7 Day Tx to EOT Day 1 Day 7 Day Tx to EOT (Pre- (on 14/15 Values (Pre- (on 14/15 Values Screen Tx) Tx) (EOT) (range) Screen Tx) Tx) (EOT) (range) Hematologic Parameters Hemolytic Parameters Hemoglobin, g/dL Indirect Bilirubin, mg/dL Pt 1 8.1 7.9 7.9 7.5 ↓ 0.4-0.6 4.7 2.8 2.5 3.0 ↓ 1.7- ↑ 0.2 Pt 2 9.2 9.9 10.4 11.1 ↑ 1.2-1.9 1.3 2.0 0.9 0.8 ↓ 0.5-1.2 Pt 3 8.7 8.1 8.8 9.2 ↑ 0.5-1.1 1.2 1.0 0.8 1.0 ↓ 0-0.2 Reticulocytes, % Lactate Dehydrogenase, U/L Pt 1 8.0 10.1 12.8 11.4 ↑ 1.3-3.4 234 180 148 192 ↓ 42- ↑ 12 Pt 2 10.2 11.0 6.8 0.8 ↓ 9.4-10.2 308 354 257 226 ↓ 82-128 Pt 3 8.0 16.0 5.8 4.2 ↓ 3.8-11.8 470 473 371 279 ↓ 191-194 EOT = end of treatment; Pre-Tx = pre-treatment; Pt = patient; SCD = sickle cell disease; Tx = treatment

Summary/Conclusion

Compound 1 has a favorable safety profile and has demonstrated PD activity after a single dose or after multiple daily doses in HS. In healthy volunteer studies, Compound 1 was well tolerated, demonstrating physiologic responses (↓ 2,3-DPG and ↑ ATP) with biologic effects including ↑ O₂ affinity, ↓ reticulocytes (P<0.001) and ↑ Hb (ns).

Compound 1 has a favorable safety profile in healthy subjects. Compound 1 demonstrates linear and time-independent PK. Reduction in 2,3-DPG and increase in ATP levels in RBCs of healthy volunteers confirms PKR activation by Compound 1. Compound 1 demonstrates proof of mechanism with increased Hb oxygen affinity in healthy volunteer RBCs, consistent with observations from in vitro mixing studies in healthy and sickle RBCs. These results support further clinical development of Compound 1, a PKR activator, in patients with SCD.

Compound 1 has a favorable safety profile in pts with SCD receiving a single dose or up to 14 days of dosing. The single dose studies in SCD subjects show an acceptable safety profile with evidence of PD activity translating into favorable biologic effects of increased oxygen affinity with a shift in the PoS to lower oxygen tensions and improved membrane deformability of sickle RBCs. Compound 1 exhibited linear and time-independent PK, leading to decreased 2,3-DPG and increased ATP levels. These results confirm that the PKR enzyme is functional and responsive to PKR activation in SCD RBCs. A single dose of Compound 1 resulted in favorable biological effects of: (1) improved oxygen affinity, decreased point of sickling and improved deformability; and (2) improved membrane function, demonstrated by an improved response to an osmotic gradient. Specifically, a single dose of Compound 1 led to decreased 2,3-DPG and increased ATP, resulting in increased O₂ affinity, decreased PoS, improved RBC deformability, and improved RBC membrane function. A single dose of Compound 1 resulted in improvements in hemoglobin, RBCs, and reticulocytes occurred when maximum PD effects were observed. These improvements indicate that a sustained 2,3-DPG reduction and increased ATP production may improve the hemolytic anemia and frequency of VOCs that characterize SCD.

Additional studies further evaluate the safety, PK/PD, and clinical activity of Compound 1 following daily administration in patients with SCD. A 2-wk SCD/MAD cohort is performed to evaluate the effects of Compound 1 on hemoglobin, inflammation and RBC metabolomics. A 12-wk dosing cohort to further characterize the effects of chronic PKR-activation on the pathophysiology of SCD is performed to evaluate the 2-wk MAD studies.

Initial blinded results of daily dosing with 300 mg Compound 1/placebo over 14 days show improvement in both hematologic and hemolytic parameters in 2 of 3 pts with SCD, along with improved RBC functional studies, suggesting the pharmacodynamic consequences of PKR activation may be of clinical benefit in SCD. Multiple-dose further evaluate the safety, PK/PD, and biological activity of Compound 1 following daily administration in pts with SCD.

Evaluation of Compound 1 for Aromatase Activity

To assess potential effects on steroidogenesis, Compound 1 was screened for steroid modulation in vitro using the H295R adreno-cortical carcinoma cell line (at 200 to 0.0002 μM) and in an assay to monitor cell viability (MTT Kit). Compound 1 indicated steroid modulation potential (% over vehicle) only at 200 M, the top concentration tested, with 100% cellular viability at concentrations ≤20 μM (90% viability at 200 μM). Based on these results, Compound 1 demonstrated no significant risk for interference with steroidogenesis considering the predicted maximum exposure (1,500 mg; C_(max) (free)=0.004 μM; AUC_(0-inf) (free)=0.002 μM·hr) of Compound 1 in human studies,

Effects on circulating levels of estradiol and testosterone in male and female healthy subjects receiving Compound 1 or placebo for a treatment period of 14 days were evaluated. Compound 1 was administered twice daily (BID) at dose levels of 100 mg, 200 mg, and 300 mg, and once daily (QD) at a dose level of 400 mg. Each dosing cohort was comprised of 9 subjects treated with Compound 1 and 3 subjects treated with placebo. Testosterone and estradiol levels were assessed prior to dosing (baseline), and then on days 8, 14 and 17. Evaluation of the change from baseline for testosterone and estradiol levels confirmed no statistically significant changes and no clinically meaningful trends, consistent with non-clinical testing indicating absence of aromatase inhibition by Compound 1.

Evaluation of Compound 1 for CYP-Mediated Activity

When evaluated for its potential towards major human CYP-mediated drug-drug interactions, Compound 1 concentrations up to 30 μM did not reversibly inhibit any of the major cytochrome P450 (CYP) isoforms in human liver microsomes (Table 27). In primary cultured hepatocytes, increases in messenger ribonucleic acid (mRNA) levels for CYP3A4, CYP1A2 and CYP2B6 at Compound 1 concentrations of 10 micromolar were low and no notable increases in mRNA (increases <2-fold) were demonstrated at Compound 1 concentrations greater than the maximum unbound clinically relevant systemic concentration and unbound inlet concentration to the liver.

Taken together, the interaction risk for Compound 1 as a CYP inducer or reversible inhibitor of concomitant medications predominantly cleared by CYP metabolism is categorized as low. Furthermore, following 14 days of dosing in healthy subjects in the clinical trial of Example 12, the observed clearance on day 1 and day 14 was unchanged, providing clinical evidence that the PK of Compound 1 is time-independent and not a substrate of auto-induction or auto-inhibition at the doses tested.

TABLE 27 Summary of IC₅₀ values of cytochrome p450 enzymes data for Compound 1 in single Substrate DDI assay IC (μM) (n = 3) Compound ID Lot # CYP1A2 CYP2C9 CYP2C19 CYP2D6 CYP3A4 Compound 1 9 >30 >30 >30 >30 >30 Furafylline 1.251 ± 0.061 Sulfsphenazole 0.863 ± 0.056 Ticlopidine 1.504 ± 0.024 Quinidine 0.0516 ± 0.00114 Ketoconazole 0.0343 ± 0.0023

TABLE 28 Fold Induction, EC₅₀ and E_(max) Values of CYP mRNA by Test Compound 1 and Positive Controls in Cultured Human Hepatocytes From Three Donors (Mean [n = 3)]) Concentrations (μM)/mRNA E_(max) Test Donor Fold Induction EC₅₀ (Fold Compound ID Isoform 0.033 0.1 0.33 1 3.3 10 (μM) Induction) Compound 1 AIH CYP1A2 0.989 0.958 1.10 1.20 1.24 1.33 N/A N/A EUJ 1.23 1.05 1.20 1.14 1.11 1.25 N/A N/A HC5-40 1.07 0.942 0.887 0.911 0.892 1.02 N/A N/A AIH CYP2B6 0.982 1.01 1.00 1.07 1.25 1.70 N/A N/A EUJ 1.17 1.23 1.10 1.29 1.26 1.43 N/A N/A HC5-40 1.17 1.06 0.964 1.00 1.24 1.21 N/A N/A AIH CYP3A4 0.940 1.13 1.11 1.41 1.84 3.60 N/A N/A EUJ 1.09 0.875 1.18 1.07 1.25 2.23 N/A N/A HC5-40 1.25 0.889 0.836 1.18 1.62 1.29 N/A N/A

Example 13: A SAD/MAD Study to Assess the Safety, Pharmacokinetics, and Pharmacodynamics of Compound 1 in Healthy Volunteers and Sickle Cell Disease Patients

Pending the results of the SAD/MAD study described in Example 12, Compound 1 can be evaluated in a registration-enabling global adaptive randomized, placebo-controlled, double blind, parallel group, multicenter trial in patients, ages 12 to 65 years, with SCD. The trial can utilize hemoglobin response as a primary endpoint while collecting additional endpoints around rates of VOC to verify clinical benefit.

Example 14: An Adaptive, Randomized, Placebo-Controlled, Double-Blind, Multi-Center Study of Oral Compound 1, a Pyruvate Kinase Activator in Patients with Sickle Cell Disease (PRAISE)

The hallmark of sickle cell disease (SCD) is hemoglobin S (HbS) polymerization upon deoxygenation, resulting in red blood cell (RBC) sickling, oxidative damage, membrane damage, hemolysis, chronic anemia, cell adhesion, vaso-occlusion and inflammation. Exacerbating the pathogenesis of SCD, the HbS RBC has increased (↑) levels of 2,3-diphosphoglycerate (2,3-DPG), resulting in reduced (↓) Hb oxygen affinity (↑ P₅₀), and reduced (↓) levels of ATP, essential for RBC homeostasis.

Compound 1 is a potent, selective, and orally bioavailable allosteric activator of erythrocyte pyruvate kinase (PKR) that increases PKR activity, resulting in reduced (↓) 2,3-DPG levels and increased (↑) ATP levels in RBCs. Preliminary data from a study in healthy volunteers and patients with SCD indicate that Compound 1 is well tolerated, has no effect on steroidogenesis, and exhibits linear and time-independent pharmacokinetics (PK) and associated pharmacodynamic (PD) responses (↓ 2,3-DPG and ↑ ATP). Furthermore, in patients with SCD, a single dose of Compound 1 demonstrated favorable biologic effects, including increased Hb oxygen affinity (↓ P₅₀), decreased point of sickling (PoS), improved RBC deformability, and improved RBC membrane function, indicative of overall improved RBC health (Example 12).

Accordingly, a phase 2/3, randomized, double-blind, placebo-controlled global study (PRAISE) was designed to investigate the safety and efficacy of Compound 1 in patients with SCD. The PRAISE study can enroll up to 344 adult and adolescent patients with SCD, including 60 to 90 patients in the Dose Determination (DD) Group and ˜274 patients in the Efficacy Continuation (EC) Group (see FIG. 56 ).

Key inclusion criteria: SCD (all genotypes), at least 2 vaso-occlusive crises (VOCs) in the past 12 mos, baseline Hb≥5.5 and ≤10 g/dL, stable hydroxyurea (HU) therapy for the previous 90 days (if applicable).

Key exclusion criteria: More than 10 VOCs in the past 12 mos, hospitalization for sickle cell crisis or other vaso-occlusive event within 14 days of consent, routine RBC transfusions, significant hepatic or renal dysfunction, history of unstable or deteriorating cardiac or pulmonary disease, or overt stroke within 2 yrs.

Endpoints: The co-primary endpoints are (1) Hb response rate at Week 24 (increase of >1 g/dL from baseline) and (2) annualized VOC rate during the blinded treatment period based on adjudicated VOC review. Secondary endpoints include measures of hemolysis, time to first VOC, and the PROMIS fatigue scale. Safety endpoints include the incidence of AEs, concomitant medications, vital signs, ECGs, clinical laboratory measurements, and physical examination.

Design: The study design is a group-sequential, adaptive, phase 2/3 study (see FIG. 56 ). Patients are stratified by age, number of VOCs (2-3 vs. 4-10) in the preceding 12 mos, and prior/concomitant HU use in the preceding 12 mos. The phase 2 DD portion assesses 2 active doses and placebo with patients randomized 1:1:1. The dose is chosen at the first interim analysis (IA1) based on safety and Hb response rate at Week 12 of the first 60 DD patients. A futility analysis is also conducted on Hb response at that point.

After dose selection, patients are randomized 1:1 into the phase 3 EC portion to assess Compound 1 efficacy. Once 110 patients from phase 2 or 3 who have been randomized to the selected dose or placebo have completed 24 weeks of follow-up or have dropped out, a second interim analysis (IA2) is performed to assess both efficacy and futility. IA2 assesses the co-primary endpoint of Hb response rate at Week 24 (p<0.001).

The final analysis after 52 weeks of blinded treatment tests the VOC endpoint, the Hb response rate, and all secondary endpoints. Key secondary endpoints are tested at IA2 and all are tested at the final analysis, when there is adequate power.

Treatment: Patients are randomized to receive Compound 1 or placebo. In the DD phase, two doses are evaluated, and in the EC phase, the selected dose of Compound 1 from the DD phase is evaluated in comparison to placebo. Patients in DD on the unselected dose remain on treatment at that dose level for 52 weeks. Following completion of 52 weeks of double-blind treatment, patients may enter a 52-week open-label extension period to receive Compound 1 at the selected dose.

Example 15: Analysis of ATP and 2,3 DPG in K2EDTA Whole Blood by LC-MS/MS

The following procedures are employed for the analysis of ATP and 2,3-DPG in human whole blood K2EDTA using a protein precipitation extraction procedure and analysis by LC-MS/MS.

This bioanalytical method applies to the parameters described below:

Assay Range 25,000-1,500,000 ng/mL Extraction Volume 15.0 μL Species/Matrix/ Water as a surrogate for Human Anticoagulant Whole Blood K2EDTA Extraction type Protein Precipitation Sample Storage 80° C. Mass Spectrometer API-5500 Acquisition software Analyst/Aria System

The following precautions are followed:

1. Standard and QC samples are prepared on ice and stored in plastic containers.

2. Study samples and QC samples are thawed on ice.

3. Extraction is performed on ice.

The following definitions and abbreviations are employed:

CRB Carryover remediation blanks FT Freeze-thaw MPA Mobile phase A MPB Mobile phase B NA Not applicable NR Needle rinse RT Retention time SIP Stability in progress TBD To be determined

The following chemicals, matrix, and reagents are used:

K₂EDTA Human Whole Blood, BioreclamationIVT or equivalent (Note: BioReclamationIVT and BioIVT are considered equivalent) Acetonitrile (ACN), HPLC Grade or better Ammonium Acetate (NH₄OAc), HPLC grade or equivalent Ammonium Hydroxide (NH₄OH, 28-30%), ACS grade or better Dimethylsulfoxide (DMSO), ACS grade or better Formic Acid (FA), 88% ACS grade Isopropanol (IPA), HPLC Grade or better Methanol (MeOH), HPLC Grade or better Water (H₂O), Milli-Q or HPLC Grade ATP-Analyte, Sponsor or supplier ATP-IS-IS, Sponsor or supplier 2,3-DPG-Analyte, Sponsor or supplier 2,3-DPG-IS-IS, Sponsor or supplier

The following procedures are used for reagent preparation. Any applicable weights and volumes listed are nominal and may be proportionally adjusted as long as the targeted composition is achieved:

Nominal Volumes Final Solution for Solution Storage Solution Composition Preparation Conditions Mobile 10 mM Weigh Ambient Phase A Ammoniumn approximately Temperature (MPA) Acetate in 770.8 mg of water pH 8.5 Ammonium Acetate; add to a bottle with 1000 mL of water. Adjust pH to 8.3-8.7 using Ammonium Hydroxide. Mobile 5:95 MPA: Add 50.0 mL of Ambient Phase B ACN MPA to 950 mL of Temperature (MPB) CAN. Mix. Needle 25:25:25: Add 500 mL of Ambient Rinse 1 25:0.1 MeOH, 500 mL of Temperature (NR1) (v:v:v:v:v) ACN, 500 mL of MeOH:ACN: H₂O, 500 mL of H₂O:IPA: IPA, and 2 mL of NH₄OH NH₄OH. Mix. Needle 90:10:0.1 Add 2 mL of FA to Ambient Rings 2 (v:v:v) 200 mL of MeOH Temperature (NR2) H₂0: and 1800 mL of MeOH:FA H₂0. Mix.

Calibration standards are prepared using water as the matrix according to the table presented below. The indicated standard is prepared by diluting the indicated spiking volume of stock solution with the indicated matrix volume.

Stock Spiking Matrix Final Final Calibration Stock Conc. Vol. Vol. Vol. Conc. Standard Solution (ng/mL) (mL) (mL) (mL) (ng/mL) STD-6 ATP Stock 60,000,000 0.0100 0.380 0.400 1,500,000 2,3-DPG Stock 60,000,000 0.0100 STD-5 STD-6 1,500,000 0.100 0.200 0.300 500,000 STD-4 STD-6 1,500,000 0.0500 0.325 0.375 200,000 STD-3 STD-6 1,500,000 0.0250 0.350 0.375 100,000 STD-2 STD-5 500,000 0.0500 0.450 0.500 50,000 STD-1 STD-5 500,000 0.0250 0.475 0.500 25,000 Cond. STD-5 500,000 0.0250 0.975 1.00 12,500

Quality control standards are prepared using water as the matrix according to the table presented below. The indicated quality control standard is prepared by diluting the indicated spiking volume of stock solution with the indicated matrix volume.

Quality Stock Spiking Matrix Final Final Control Conc. Vol. Vol. Vol. Conc. Standard Stock Solution (ng/mL) (mL) (mL) (mL) (ng/mL) QC-High ATP Stock 60,000,000 0.160 7.68 8.00 1,200,000 2,3-DPG Stock 60,000,000 0.160 QC-Mid QC-High 1,200,000 1.50 4.50 6.00 300,000 QC-Low QC-Mid 300,000 1.50 4.50 6.00 75,000

An internal standard spiking solution is prepared with a final concentration of 12,500 ng/mL ATP and 2,3-DPG by diluting stock solutions of ATP and 2,3-DPG at concentrations of 1,000,000 ng/mL with water. 0.200 mL each of the ATP and 2,3-DPG stock solutions are diluted with 15.6 mL of water to produce a final volume of 16.0 mL at a final concentration of 12,500 ng/mL of ATP and 2,3-DPG.

The following procedures are used for sample extraction prior to analysis via LC-MS/MS. 15.0 μL of the calibration standards, quality controls, matrix blanks, and samples are aliquoted into a 96-well plate. 50.0 μL of the internal standard spiking solution is added to all samples on the plate, with the exception of the matrix blank samples; 50.0 μL of water is added to the matrix blank samples. Subsequently, 150 μL of water is added to all samples on the plate. The plate is then covered and agitated by vortex at high speed for ten minutes, after which 750 μL of methanol is added to all samples on the plate. The plate is covered and agitated by vortex for approximately 1 minute. The plate is then centrifuged at approximately 3500 RPM at approximately 4° C. for five minutes. After centrifugation, a liquid handler is used to transfer 50 μL of each sample to a new 96-well plate, and 200 μL of acetonitrile is added to all samples on the plate. The newly prepared plate is covered and agitated by vortex for approximately 1 minute. The plate is then centrifuged at approximately 3500 RPM at approximately 4° C. for 2 minutes.

The following LC parameters and gradient conditions are used for analysis of the extracted samples:

LC Parameters Analytical Vendor: SeQuant Column Description: ZIC-pHILIC Dimensions: 50 mm × 2.1 mm Column Heater Temperature: 40° C. Plate Position: Cold Stack Rack Cold Stack Set Point:  5° C. Mobile Mobile Phase A 10 mM Ammoniumn Phase (MPA) Acetate in water pH 8.5 Mobile Phase B 5:95 MPA:ACN (MPB) Injection 5 μL Volume

LC Gradient Time Flow Gradient Step (s) (mL/min) Setting % MPB 1 50 0.400 Step 5 2 30 0.400 Ramp 95 3 70 0.400 Step 5 Data is collected starting at 0.08 min and is collected over a data window length of 0.70 min.

The following MS parameters are used for analysis of the extracted samples using an API-5500 Mass Spectrometer:

Interface: Turbo Ion Spray Ionization, positive-ion mode Scan Mode: Multiple Reaction Monitoring (MRM) Scan Parameters: Parent/Product: Dwell Time (ms): 506.0/159.0 50 521.0/159.0 25 265.0/166.8 50 268.0/169.8 25 Source 400° C. Temperature:

Example 16: Measuring Oxygen Affinity (p50)

Oxygen reversibly binds to the heme portions of the Hgb molecule. As oxygenated blood flows via capillaries to peripheral tissues and organs that are actively consuming oxygen, PO₂ drops and Hgb releases oxygen. The affinity of oxygen for hemoglobin can be measured in a sigmoidal oxygen equilibrium curve. In the scan, the Y-axis plots the percent of hemoglobin oxygenation and the X-axis plots the partial pressure of oxygen in millimeters of mercury (mm Hg). If a horizontal line is drawn from the 50% oxygen saturation point to the scanned curve and a vertical line is drawn from the intersection point of the horizontal line with the curve to the partial pressure X-axis, a value commonly known as the p50 is determined (i.e., this is the pressure in mm Hg when the scanned hemoglobin sample is 50% saturated with oxygen). This relationship can be impacted by temperature, pH, carbon dioxide, and the glycolytic intermediate 2,3-DPG. 2,3-DPG binds within the central cavity of the Hgb tetramer, causes allosteric changes, and reduces Hgb's affinity for oxygen. Under physiological conditions (i.e., 37° C., pH=7.4, and partial carbon dioxide pressure of 40 mm Hg), the p50 value for normal adult hemoglobin (HbA) is around 26.5 mm Hg. If a lower than normal p50 value is obtained for the hemoglobin under test, the scanned curve is considered to be “left-shifted” and the presence of high affinity hemoglobin is indicated. If a higher than normal p50 value is obtained for the hemoglobin under test, the scanned curve is considered to be “right-shifted” and the presence of low affinity hemoglobin is indicated.

The oxygen affinity of RBCs was measured in patient blood using a Hemox Analyzer (TCS Scientific Corp.), an automatic system for the recording of blood oxygen equilibrium curves and related phenomena. The Hemox Analyzer was used according to standard methods to determine the hemoglobin-oxygen dissociation curves for whole blood samples, numerically characterized by the p50, the partial pressure of oxygen at which hemoglobin is 50% saturated. The operating principle of the Hemox-Analyzer is based on dual-wavelength spectrophotometry for the measurement of the optical properties of hemoglobin and a Clark electrode for measuring the oxygen partial pressure in millimeters of mercury. Whole blood is diluted and placed into a special plastic cuvette that is maintained at 37° C. To perform the analysis, a beam of polychromatic light is passed through the cuvette and is made monochromatic prior to reaching the photomultiplier detectors. In the case of hemoglobin, the wavelength of maximum absorbance is the measuring wavelength (560 nm), while the reference wavelength is at the isosbestic point at (570 nm). The absorbance at the isosbestic point remains unchanged during the deoxygenation process of the hemoglobin, however the measuring wavelength (560 nm) undergoes a drastic change in absorbance. This change is detected by the electronic circuitry and is plotted as the log/ratio change between the two wavelengths. The log/ratio measurement at 560 nm and 570 nm is utilized to measure the optical absorbance change during the deoxygenation of the hemoglobin. Simultaneously with the measurement of the hemoglobin absorbance, the oxygen concentration is directly measured in the sample using a Clark electrode. Under normal atmospheric conditions of 760 mm of mercury the oxygen concentration (i.e., the oxygen partial pressure) is 149 mm of mercury. This saturation point is used for full-scale calibration of the computer prior to starting the plotting of the curve. When the oxygen is being replaced by an inert gas (nitrogen) in a continuous procedure, hemoglobin becomes deoxygenated.

Blood samples for testing were obtained and handled as follows. Specimen samples of 3 mL of whole blood are collected in tubes containing EDTA (Lavender). A minimum volume of 500 μL of whole blood is required. Blood collected in Sodium or lithium heparin are acceptable, but EDTA is the preferred anti-coagulant. A control sample drawn from a healthy normal volunteer must be processed with each patient sample. The normal control should be handled in the same manner as patient sample (i.e., date of draw, anti-coagulant used, sample storage conditions). Store all specimens at 2-8° C. upon receipt in the laboratory. Specimens must be shipped overnight with a cold pack to maintain shipping temperature ˜4° C. and be accompanied by a normal control. Samples are stable in EDTA anti-coagulated blood held at 2-8° C. for 48 hours. Any clotted samples, samples stored in suboptimal conditions, or samples with less than 200 uL volume and samples greater than 48 hours old are rejected.

The following references provide additional guidance on the method of obtaining oxygen affinity curves and determination of p50 as described above:

-   1. Operation Manual for the Hemox-Analyzer, TCS Scientific, New     Hope, Pa., revised Jan. 10, 2007. -   2. Ellis S S, Pepple D J. Sildenafil Increases the p50 and Shifts     the Oxygen-Hemoglobin Dissociation Curve to the Right. J Sex Med.     2015; 12(12):2229-32. doi: 10.1111/jsm.13038. -   3. McKoy M, Allen K, Richards A, Pepple D. Effect of cilostazol on     the p50 of the oxygen-hemoglobin dissociation curve. Int J Angiol.     2015; 24(1):67-70. doi: 10.1055/s-0034-1383433. -   4. Guarnone R, Centenara E, Barosi G. Performance characteristics of     Hemox-Analyzer for assessment of the hemoglobin dissociation curve.     Haematologica. 1995 September-October; 80(5):426-30. -   5. Vanhille D L, Nussenzveig R H, Glezos C, Perkins S, Agarwal A M.     Best practices for use of the HEMOX analyzer in the clinical     laboratory: quality control determination and choice of     anticoagulant. Lab Hematol. 2012; 18(3):17-9.

Example 17: Oral Bioavailability of Compound 1 Pharmaceutical Compositions

The systemic exposure of Compound 1 in rats and mice was evaluated by dosing a spray dried dispersion (SDD) obtained from Step 6 of Example 1, containing Compound 1 and HPMC AS-MG (1:3) dispersed in an aqueous vehicle (0.5% Hydroxypropylmethyl Cellulose in water).

For comparison, a crystalline form (designated Type A) of Compound 1 was also prepared and characterized. Type A was characterized by XRPD (Method A), TGA, DSC, and DVS analysis.

Method A. XRPD analysis was performed with a Panalytical X'Pert3 Powder XRPD on a Si zero-background holder. The 20 position was calibrated against Panalytical 640 Si powder standard. Details of the XRPD method used in the experiments are listed in the Table below.

Parameters for Reflection Mode X-Ray wavelength Cu, kα, Kα1 (Å): 1.540598, Kα2 (Å): 1.544426 Kα2/Kα1 intensity ratio: 0.50 X-Ray tube setting 45 kV, 40 mA Divergence slit Automatic Scan mode Continuous Scan range (°2TH) 3°-40° Step size (°2TH) 0.0262606 Scan speed (°/s) 0.066482

The XRPD pattern for Compound 1 solid form Type A obtained by Method A above was characterized by the XRPD 2-theta peaks and d-spacing summarized in the following table:

Pos. [°2Th.] d-spacing [Å] 4.61 19.19 5.80 15.24 7.22 12.25 7.68 11.50 11.21 7.89 12.31 7.19 14.44 6.13 15.66 5.66 16.95 5.23 18.02 4.92 19.20 4.62 20.48 4.34 21.35 4.16 21.66 4.10 22.47 3.96 23.19 3.84 24.76 3.60 26.73 3.34 28.01 3.19 28.49 3.13 29.35 3.04 30.25 2.95 32.14 2.79 34.12 2.63 36.46 2.46

The TGA and DSC curves for solid form Type A of Compound 1 showed 1.9% weight loss up to 100° C. by TGA and two endotherms at 85.9° C. (peak temperature) and 146.0° C. (onset temperature) by DSC. Type A was analyzed by DSC by heating to 120° C. and cooled to 25° C., then heated up to 300° C. No endotherm below 100° C. was observed in the second heating cycle. XRPD analysis after DSC cycling showed no form change compared to Type A. DVS results of Type A of Compound 1 showed a 3.4% water uptake up to 40% RH (ambient condition), and 1.0% water uptake from 40% RH to 80% RH at RT, indicating that Type A is hygroscopic. No form change was observed for Type A before and after DVS test at RT, as determined by XRPD. Based on the foregoing analytical data, Type A is believed to be a channel hydrate.

The SDD formulation (“500 mpk SDD”) dosed at 500 mg/kg to rats showed an AUC_(last) that was 40× greater than the maximum exposure obtained with the standard formulation (“300 mpk Suspension” made up of Compound 1 (Type A) in 10% Propylene Glycol, 10% Cremophore, 80% Water), as shown in the data in the Table below. Additionally, the exposure of a 500 mpk Nano-Suspension made up of nanoparticles of Compound 1 (Type A) was evaluated. Robust exposure was observed with SDD formulation in mouse as well. Results are shown in FIG. 57 .

t_(1/2) t_(max) C_(max) AUClast Animal (h) (h) (ng/mL) (h*ng/mL) Rat 3.22 1.67 44400 180603 Mouse 2.54 0.5 75200 113369

Several formulation compositions of Compound 1, including an SDD made up of Compound 1 and HPMC AS-MG (1:3), were evaluated in monkeys. The compositions of the tested oral dosage formulations are listed in the Table below; Compound 1 exposure results for each formulation are shown in FIG. 58 .

Formulation Dosage Form Composition Formulation #1 Capsule; Size 0 Compound 1 (Type A), (with Bile Salt) White Opaque micronized 49.9% Gelatin Avicel PH101 23.5% AcDiSol 5.0% SLS 10.1% Na Taurocholate 10.0% Mg Stearate 0.5% Silicon Dioxide 1.0% Formulation #2 Capsule; Size 0 Compound 1 (Type A) (Formulated White Opaque micronized API 49.9% Capsule) Gelatin Avicel PH101 33.3% AcDiSol 5.0% SLS 10.3% Mg Stearate 0.5% SiO2 1.0% Formulation #3 Capsule; Size 0 Compound 1 (Type A) (Micronized fill) White Opaque micronized API only Gelatin Formulation #4 Suspension Compound 1 Spray (SDD) Dried Dispersion 0.5% Hydroxypropylmethyl Cellulose in Water

The formulations were evaluated for pharmacokinetic parameters in monkeys and are shown in FIG. 58 . The profiles show that the SDD formulation (Formulation 4) provided a significant enhancement in overall exposure compared to the encapsulated formulations (Formulations 1, 2, and 3). The bioavailability enhancement with the SDD formulation is approximately 50-62%, which is several fold higher compared to the other formulations, at a dose equivalent to 100 mg. 

We claim:
 1. The compound (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one for use in a single daily (QD) administration to increase hemoglobin oxygen affinity in the red blood cells (RBCs) of a human subject as measured by a reduced p50 (pO₂ at 50% hemoglobin saturation) in the RBCs at 24 hours after the administration of the compound.
 2. The compound of claim 1, for use in daily (QD) administration for 14 consecutive days to increase hemoglobin oxygen affinity in the red blood cells (RBCs) of a human subject as measured by a reduced p50 (pO₂ at 50% hemoglobin saturation) measured in the RBCs at after 14 days of QD administration of the compound to the human subject.
 3. The compound of claim 1, for use in reducing the 2,3-DPG concentration in the blood of the human subject by at least 30% at 24 hours after the administration of the compound.
 4. The compound of claim 1, for use in increasing the ATP concentration in the blood of the human subject by at least 40% after administering the compound once daily to the subject for 14 consecutive days.
 5. The compound of claim 1, for use in simultaneously activating PKR, increasing ATP, decreasing 2,3-DPG and increasing oxygen affinity (p50) in the blood of the subject for 72 hours after administering the compound to the subject.
 6. The compound of any one of claims 1-5, wherein the human subject is diagnosed with Sickle Cell Disease (SCD).
 7. The compound of claim 6, wherein the pediatric SCD patient is at least age
 12. 8. The compound of claim 1, wherein the human subject is at least age
 18. 9. The compound of claim 1, wherein the human subject is diagnosed one of the following hemoglobin genotypes: Hgb SS, Hgb Sβ⁺-thalassemia, Hgb Sβ⁰-thalassemia, or Hgb SC.
 10. The compound (S)-1-(5-((2,3-dihydro-[1,4]dioxino[2,3-b]pyridin-7-yl)sulfonyl)-3,4,5,6-tetrahydropyrrolo[3,4-c]pyrrol-2(1H)-yl)-3-hydroxy-2-phenylpropan-1-one for use in the treatment of Sickle Cell Disease in a human subject having a Hgb SS or Hgb SC hemoglobin genotype. 